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Long-term toxicity to fish

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Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 5, 2019 to December 5, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals, 210 Fish, early-life stage toxicity test. Paris: OECD, 26 July 2013.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: National Guideline
Version / remarks:
State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, 210 Fish, Early-life Stage Toxicity Test [M]. Second Edition. Beijing: China Environmental Science Press, 2013:86-93.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: National Guideline
Version / remarks:
China National Technical Committee for Standardization of Dangerous Chemical Management GB/T 21854-2008.Chemical Fish, Early-life Stage Toxicity Test [S]. Beijing: China Standardization Press, 2008.
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
No further details specified in the study report.
Analytical monitoring:
yes
Details on sampling:
The actual concentrations of test substance in 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were measured at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d, 33 d (new solution) and 1 d, 8 d, 11 d, 15 d, 22 d, 28 d, 34 d (old solution).
Vehicle:
no
Details on test solutions:
The test substance is a kind of UVCB item, and insoluble in test water, according to testing guidance water accommodate fraction was prepared as test solution. The preparation method is shown as below:
1d prior to the test – 8 d: 5.0 mg, 16 mg, 50 mg, 160 mg and 500 mg test substance were weighed into five flasks and mixed with small amount of dechlorinated tap water, then the mixtures were transferred into 5 L vessels and diluted with dechlorinated tap water to 5000 mL respectively to obtain the nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L. The solutions were stirred for about 24 h (the depth of vortex was maintained at one third of the test solution height) and stood for about 1 h, then 2000 mL of middle layer solutions were collected into another 5 L vessels (the first 100 – 200 mL solution was discarded), the nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF solutions were obtained and divided into three replicates as test solutions ( 200 mL / replicate).
9 d – 32 d: 10 mg, 32 mg, 100 mg, 320 mg and 1000 mg test substance were weighed into five flasks and mixed with small amount of dechlorinated tap water, then the mixtures were transferred into 10 L vessels and diluted with dechlorinated tap water to 10000 mL respectively to obtain the nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L suspensions. The suspensions were stirred for about 24 h (the depth of vortex was maintained at one third of the test solution height) and stood for about 1 h, then 7000 mL of middle layer solutions were collected into another 10 L vessels (the first 100 – 200 mL solution was discarded), the nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF solutions were obtained and divided into three replicates as test solutions (2000 mL / replicate).
Note: After stirring, microscopic inspection was used to check whether the micro-dispersions or insoluble test material present in the test solution, the solution that collected at 0 d, 7 d, 14 d, 21 d, 28 d and 32 d were observed.
Test organisms (species):
other: Gobiocypris rarus
Details on test organisms:
(1) Species: Rare Minnow(Gobiocypris rarus)
(2) Reason for selection of species: The test species is recommended by “The Guidelines for the Testing of Chemicals, 210 Fish, Early-life Stage Toxicity Test” (Chemical Registration Center of MEP, Second Edition).
(3) Supplier: The brood fish with batch number FGr20181031-1 were provided by Safety Evaluation Center of Shenyang Chemical Research Institute CO. LTD.
(4) The standard of embryo: The embryos should not older than 8 h after fertilized
(5) Obtained embryo: The brood fish were hold in the water maintained the temperature at 25 °C ± 2 °C with 14 h light /10 h darkness. Before the test, the female and male was paired as 1:1, the embryos collected by artificial fertilisation.
(6) Selection and treatment of embryo: Embryos with normal development and good morphology were selected under an anatomical microscope. The normal division embryos are sticky and transparent. However, unfertilized embryos are observed as milky white.
(7) Number of embryo: 360 embryos
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
34 d
Post exposure observation period:
No post exposure observation period specified in the study report.
Hardness:
At the start of the test, the total hardness of the blank control and 100 mg/L WAF test substance group were both 110 mg/L.
Test temperature:
The temperature of all test vessels measured regularly ranged from 24.1 °C to 25.5 °C, measured continuously ranged from 24.0 °C to 26.0 °C.
pH:
During the test, the pH of the test solution was ranged from 7.51 to 8.40.
Dissolved oxygen:
The dissolved oxygen concentration ranged from 61% to 99% of the air saturation value (ASV) and met the requirement of at least 60% of ASV.
Salinity:
The salinity of the blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were all 0‰.
Conductivity:
Not specified
Nominal and measured concentrations:
The measured concentrations of test substance in blank control at 0 d and 1 d (old solution) were both lower than LOD of 0.645 mg/L.
The measured concentrations of test substance in nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d, 33 d (new solution) and 1 d, 8 d, 11 d, 15 d, 22 d, 28 d, 34 d (old solution) were all lower than LOD of 0.645 mg/L
Details on test conditions:
Test water
Dechlorinated tap water was used in the test. Before the test, the test water was dechlorinated by aeration so that the dissolved oxygen concentration reached more than 80% of the air saturation value.

Design of the difinitive test
According to the results of preliminary test (see Annex C), cumulative abnormal rates of blank control and 10 mg/L WAF and 100 mg/L WAF test groups were all 0%. No abnormal appearances and behaviors were observed during the test. The measured concentrations of test substance were all lower than the limit of detection, so the stability of test substance could not be confirmed. Therefore, five nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L water accommodated fraction (WAF) test groups with a geometric factor of 3.2 and one blank control were set in the definitive test, with 20 embryos for each replicate and three replicates for each test group.

Test conditions
Conditions of exposure
(1) Type of the test
The limit test was conducted as semi-static test with renew at least 3/4 volume test solution every 24 h (As the embryos were obtained at night of 0 d, in order to perform the test expediently, the time of the first renewal solution was performed on about 16 h after the start of the exposure).

(2) Exposure duration
34 d

(3) Test concentration
Five nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L water accommodated fraction (WAF) test groups with a geometric factor of 3.2 and one blank control were set in the definitive test.

(4) Control
A blank control (test water without test substance) was set.

(5) Replicate
3 replicates/test group

(6) Numbers of embryos
20 embryos/test vessel, 60 embryos/test group, 360 embryos in total

(7) Volume of test solution
0 d – 9 d: about 200 mL / test vessel, 10 d- 34 d: about 2000 mL/ test vessel.

Conditions of test environment
(1) Water temperature
The temperature of all test vessels measured regularly ranged from 24.1 °C to 25.5 °C, and measured continuously ranged from 24.0 °C to 26.0 °C.

(2) Dissolved oxygen concentration
The dissolved oxygen concentration in the test solutions was kept at least 60% of the air saturation value (ASV) throughout the test.

(3) pH
The test was performed without adjustment of pH and the pH was in the range of 7.51 to 8.40 during the test period.

(4) Light
Artificial light of white fluorescent lamp was used. The photoperiod was 14 h light / 10 h darkness.

Observation and measurement
(1) Observation embryos and larvae or juvenile fish
Stage of embryonic development: The embryonic stages were recorded at the start of exposure time.
Hatching and survival: Observations on hatching and survival were made daily and numbers recorded, removed dead individuals.
Abnormal appearance: The numbers of embryos or larvae or juvenile fish showing abnormality of body form were recorded daily.
Abnormal behaviour: The behavour of larvae or juvenile fish after hatched were observed and recorded daily.

(2) Temperature, pH and Dissolved oxygen concentration
The temperature, pH and dissolved oxygen of all test vessels at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d , 33 d (new solution) and 1 d, 8 d, 15 d , 22 d, 28 d, 34 d (old solution). Meanwhile, the temperature of incubator or test environment water were continuously monitored during the test.

(3) Total hardness of test solution
Total hardness of blank control and 100 mg/L WAF test group were measured at the beginning of the test.

(4) Salinity of test solution
The salinity of the blank control and all test groups were measured at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d , 33 d (new solution) and 1 d, 8 d, 15 d , 22 d, 28 d, 34 d (old solution).

(5) Measurement of individual lengths
Total lengths of surviving larvae or juvenile fish were measured at the end of the test.

(6) Measurement the dry weights of larvae or juvenile fish
At the end of the test, all surviving test larvae in each replicate were taken into a culture dish and dried (24 hours at 60 ºC), the culture dish were dried at 105°C for 2 hours prior to use.

(7) Measured concentrations of test substance in test solutions
The actual concentrations of test substance in 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were measured at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d, 33 d (new solution) and 1 d, 8 d, 11 d, 15 d, 22 d, 28 d, 34 d (old solution) by GC; the blank control was measured at 0 d (new solution) and 1 d (old solution).

Data process
Cumulative mortality, numbers of healthy larvae, numbers of hatching larvae per day, lengths and weights of surviving fish, numbers of larvae or juvenile fish that are deformed or abnormal appearance, numbers of larvae or juvenile fish exhibiting abnormal behaviour were statistical. Alternatively, obtained the NOEL and LOEL based on the total length was estimated by SPSS 20.0 with methods of the normal distribution, variance of homogeneity and Jonckneere-Terpstra Test; obtained the NOEL and LOEL based on the dry weight was estimated by SPSS 20.0 with methods of the normal distribution, variance of homogeneity and Dunnett t-Tests; obtained the NOEL and LOEL based on the surviving fish and the cumulative abnormality with the method of Chi-Square Test.
Reference substance (positive control):
not required
Key result
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
number hatched
Key result
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Key result
Duration:
34 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
34 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
number hatched
Key result
Duration:
34 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
34 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Details on results:
Observation of test solutions
The WAF solution was prepared with the direct addition method. During the preparation periods, surface films in the top layers were observed in all test solutions, so the solution in the middle layer was siphoned into another test vessel as exposure solution, certainly, there no obvious droplets or surface films were observed in the exposure solution. Any remaining non-dissolved test chemical component in the WAF has the possibility to cause physical effects on the test organism unrelated to inherent chemical toxicity.

Hatching rate, surviving rate after post-hatched, cumulative mortality and cumulative abnormal rate of embryos / larvae or juvenile fish
The blastocyst stage of embryos that not older than 8 h after fertilized were exposed. All embryos were completely hatched at 4th day after start of the test, the hatching rates of embryos in blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were both 100%. The hatching rate of blank control had met the validity criterion that the hatched success should greater than 70%.

The abnormal appearances of embryos and larvae or juvenile fish
During the test, no abnormal appearances were observed in blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups .

The abnormal behaviors of larvae or juvenile fish
During the test, no abnormal behaviors were observed in the blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups .

The total lengths of surviving larvae or juvenile fish
At the end of the test, the mean and the standard deviation of total length of surviving larvae or juvenile fish in blank control was 17.23 mm ± 0.21 mm , coefficient of variation (CV) was 1.24%, which met the validity criterion that the CV should not exceed 20%.
The mean and standard deviation of total length of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were 17.47 mm ± 0.08 mm, 17.87 mm ± 0.08 mm, 17.60 mm ± 0.49 mm, 17.99 mm ± 0.27 mm and 17.76 mm ± 0.21 mm, respectively. The mean total lengths of all test groups were greater than the blank control’s, based on the total lengths, there no obvious toxicity effect on the larvae or juvenile fish, so the NOEL was the loading rate of 100 mg/L, and the LOEL was greater than the loading rate of 100 mg/L .

The dry weights of surviving larvae or juvenile fish
The results of dry weight of surviving larvae or juvenile fish are shown in Table 5.
At the end of the test, the average and standard deviation of dry weight of surviving larvae or juvenile fish in blank control was 10.96 mg±0.34 mg , the coefficient of variation was 3.08%, which was met the validity criterion that the coefficient of variation should not exceed 20%.

The temperature, pH, dissolved oxygen, hardness and salinity
During the test, the pH of the test solution was ranged from 7.51 to 8.40. The temperature of all test vessels measured regularly ranged from 24.1 °C to 25.5 °C, and measured continuously ranged from 24.0 °C to 26.0 °C, the temperature was not differ by more than ±1.5 °C between test chambers or between successive days at any time during the test, and the temperature was within the range of 25°C  1°C. The dissolved oxygen concentration ranged from 61% to 99% of the air saturation value (ASV) and met the requirement of at least 60% of ASV.
At the start of the test, the total hardness of the blank control and 100 mg/L WAF test substance group were both 110 mg/L, and the salinity of the blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were all 0‰.

Analytical concentration of test substance in test solution
Sample pretreatment and preservation
The test solutions were collected and filtered through 0.45 μm aqueous phase PES filter membranes (the first 5 mL - 10 mL filtrate was discarded), 50 mL filtrate was transferred into a separating funnel and extract three times with 20 mL, 15 mL and 10 mL of n-hexane, respectively. The three extracts were collected into a 50 mL volume flask and diluted to 50 mL with n-hexane before analyzed. The samples were analyzed immediately, otherwise the samples were stored at 2 °C – 8 °C and protected from light after pretreatment, and the measurement were carried out within 96 h.

Measured concentrations of test substance
The limit of detection (LOD) was 0.645 mg/L and limit of quantification (LOQ) was 2.15 mg/L.
The measured concentrations of test substance in blank control at 0 d and 1 d (old solution) were both lower than LOD of 0.645 mg/L.
The measured concentrations of test substance in nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d, 33 d (new solution) and 1 d, 8 d, 11 d, 15 d, 22 d, 28 d, 34 d (old solution) were all lower than LOD of 0.645 mg/L. The measured concentrations of all test solutions lower than the LOD, this did not mean that there was no test substance present in the test solutions, just that the concentration was below the LOD of the analytical method, indicating that test substance was poorly soluble in test water.
Results with reference substance (positive control):
Not required
Reported statistics and error estimates:
At the end of test, the surviving rate after post-hatched of blank control was 97%, which met the validity criterion that the surviving rate after post –hatched should not less than 75%. The surviving rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were 95%, 97%, 93%, 97% and 93%, respectively. The method of Chi-Square Test was used based on the surviving rate after post-hatched, and the statistical result showed that the NOEL was 100 mg/L WAF, and the LOEL was greater than 100 mg/L WAF.
At the end of test, the cumulative mortalities of embryos / larvae or juvenile fish in blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were 3%, 5%, 3% , 7% , 3% and 7% , respectively. The method of Chi-Square Test was used based on the cumulatively mortality, and the statistical result showed that the NOEL was 100 mg/L and the LOEL was greater than 100mg/L.
At the end of test, the cumulative abnormal rates of embryos / larvae or juvenile fish in blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were both 0%. The method of Chi-Square Test was used based on cumulative abnormal rates and the statistical result showed that the NOEL was 100 mg/L WAF, and the LOEL was greater than 100 mg/L WAF.
The means and standard deviation of dry weights in 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were 11.40 mg ± 0.28 mg,11.78 mg ± 0.30 mg, 11.65 mg ± 0.91 mg, 12.59 mg ± 0.85 mg and 12.06 mg ± 0.67 mg, respectively. Results of Dunnett t-test showed that, there was no statistically significant difference (p>0.05) based on dry weights between the blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups , so based on the dry weight of surviving larvae or juvenile fish, the NOEL was the loading rate of 100 mg/L and the LOEL was greater than loading rate of 100 mg/L.

Hatching rate, surviving rate after post-hatch, cumulative mortality and cumulative abnormal rate of embryos/larvae at the end of test

The normal loading rate

Replicate

 

Total embryos

Number of hatching

Hatching rate

Mean of hatching rate

Number of abnormal

Total abnormality rate

Mean of abnormality rate

Number of deaths

Mortality rate

Mean of mortality

Surviving fish after post-hatch

Surviving rate

Mean of surviving rate

(individual)

(tail)

(%)

M±SD

(individual)

(%)

M±SD

(individual)

(%)

M±SD

(individual)

(%)

M±SD

Blank control

1

20

20

100

100±0

0

0

0±0

1

5

3±3

19

95

97±3

2

20

20

100

0

0

0

0

20

100

3

20

20

100

0

0

1

5

19

95

1.0 mg/L WAF

1

20

20

100

100±0

0

0

0±0

0

0

5±5

20

100

95±5

2

20

20

100

0

0

1

5

19

95

3

20

20

100

0

0

2

10

18

90

3.2 mg/L WAF

1

20

20

100

100±0

0

0

0±0

0

0

3±6

20

100

97±6

2

20

20

100

0

0

2

10

18

90

3

20

20

100

0

0

0

0

20

100

10 mg/L WAF

1

20

20

100

100±0

0

0

0±0

0

0

7±6

20

100

93±6

2

20

20

100

0

0

2

10

18

90

3

20

20

100

0

0

2

10

18

90

32 mg/L WAF

1

20

20

100

100±0

0

0

0±0

0

0

3±3

20

100

97±3

2

20

20

100

0

0

1

5

19

95

3

20

20

100

0

0

1

5

19

95

100 mg/L WAF

1

20

20

100

100±0

0

0

0±0

2

10

7±6

18

90

93±6

2

20

20

100

0

0

0

0

20

100

3

20

20

100

0

0

2

10

18

90

Note: M: Mean; SD: Standard Deviation 

 

Observation of hatching, abnormal appearance and mortality

The normal loading rate

Replicate

Observation (light: number of hatching / number of surviving; right: type of appearance (number) / type of death (number))

1d

2d

3d

4d

Blank control

1

0/20

N

0/20

N

0/20

N

0/20

N

2

0/20

N

0/20

N

0/20

N

0/20

N

3

0/20

N

0/20

N

0/20

N

0/20

N

1.0 mg/L WAF

1

0/20

N

0/20

N

0/20

N

0/20

N

2

0/20

N

0/20

N

0/20

N

0/20

N

3

0/20

N

0/20

N

0/20

N

0/20

N

3.2 mg/L WAF

1

0/20

N

0/20

N

0/20

N

0/20

N

2

0/20

N

0/20

N

0/20

N

0/20

N

3

0/20

N

0/20

N

0/20

N

0/20

N

10 mg/L WAF

1

0/20

N

0/20

N

0/20

N

0/20

N

2

0/20

N

0/20

N

0/20

N

0/20

N

3

0/20

N

0/20

N

0/20

N

0/20

N

32 mg/L WAF

1

0/20

N

0/20

N

0/20

N

0/20

N

2

0/20

N

0/20

N

0/20

N

0/20

N

3

0/20

N

0/20

N

0/20

N

0/20

N

100 mg/L WAF

1

0/20

N

0/20

N

0/20

N

0/20

N

2

0/20

N

0/20

N

0/20

N

0/20

N

3

0/20

N

0/20

N

0/20

N

0/20

N

Note: “N” means normal

 

The abnormal behaviours of larvae or juvenile fish during the test

The normal loading rate

Replicate

Observations (left: appearance /death /abnormal behaviour /surviving; right: type of abnormal appearance (number) /type of death (number) /abnormal behaviour (number))

4d

5d

6d

7d

8d

9d

Blank control

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

3

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

1.0 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

3

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

3.2 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

3

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

10 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

3

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

32 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

3

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

100 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

3

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

Note: “N” means normal. The abnormal behaviours were observed after post-hatched.

The abnormal behaviours of larvae or juvenile fish during the test (continuation)

The normal loading rate

Replicate

Observations (left: appearance /death /abnormal behaviour /surviving; right: type of abnormal appearance (number) /type of death (number) /abnormal behaviour (number))

10d

11d

12d

13d

14d

15d

Blank control

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

3

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/1/0/19

IMM(1)

1.0 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/1/0/19

IMM(1)

3

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/1/0/19

IMM(1)

3.2 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

3

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

10 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/1/0/19

IMM(1)

0/0/0/20

N

3

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/1/0/19

IMM(1)

0/0/0/20

N

32 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

3

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/1/0/19

IMM(1)

100 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/2/0/18

IMM(2)

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

3

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/1/0/19

IMM(1)

0/1/0/18

IMM(1)

Note: “N” means normal. The abnormal behaviours were observed after post-hatched. “IMM” mean immobile, and the fish were dead.

The abnormal behaviours of larvae or juvenile fish during the test (continuation)

The normal loading rate

Replicate

Observations (left: appearance /death /abnormal behaviour /surviving; right: type of abnormal appearance (number) /type of death (number) /abnormal behaviour (number))

16d

17d

18d

19d

20d

21d

Blank control

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/1/0/19

IMM(1)

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

3

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

1.0 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

3

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/1/0/18

IMM(1)

0/0/0/18

N

3.2 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/1/0/19

IMM(1)

0/0/0/19

N

3

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

10 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/1/0/18

IMM(1)

0/0/0/18

N

3

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/1/0/18

IMM(1)

0/0/0/18

N

32 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/1/0/19

IMM(1)

3

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

100 mg/L WAF

1

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

3

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

Note: “N” means normal. The abnormal behaviours were observed after post-hatched. “IMM” mean immobile, and the fish were dead.

The abnormal behaviours of larvae or juvenile fish during the test (continuation)

The normal loading rate

Replicate

Observations (left: appearance /death /abnormal behaviour /surviving; right: type of abnormal appearance (number) /type of death (number) /abnormal behaviour (number))

22d

23d

24d

25d

26d

27d

Blank control

1

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

3

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

1.0 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

3

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

3.2 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/19

N

0/1/0/18

IMM(1)

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

3

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

10 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

3

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

32 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

3

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

100 mg/L WAF

1

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

3

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

Note: “N” means normal. The abnormal behaviours were observed after post-hatched. “IMM” mean immobile, and the fish were dead.

The abnormal behaviours of larvae or juvenile fish during the test (continuation)

The normal loading rate

Replicate

Observations (left: appearance /death /abnormal behaviour /surviving; right: type of abnormal appearance (number) /type of death (number) /abnormal behaviour (number))

28d

29d

30d

31d

32d

33d

34d

Blank control

1

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

3

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

1.0 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

3

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

3.2 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/19

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

3

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

10 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

3

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

32 mg/L WAF

1

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

2

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

3

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

0/0/0/19

N

100 mg/L WAF

1

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

2

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

0/0/0/20

N

3

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

0/0/0/18

N

Note: “N” means normal. The abnormal behaviours were observed after post-hatched. “IMM” mean immobile, and the fish were dead.

 

The total length of larvae or juvenile fish at the end of the test

The nominal loading rate

Replicate

Total length (mm)

1

2

3

4

5

6

7

8

9

10

11

12

Blank control

1

17.73

16.96

16.96

18.35

16.57

16.74

17.28

17.89

16.96

16.91

18.60

16.75

2

17.38

16.37

18.59

18.25

15.71

16.76

17.81

17.61

18.59

17.84

16.47

16.48

3

16.62

16.39

17.28

17.76

18.05

16.65

15.31

15.42

17.40

17.67

17.97

17.07

1.0 mg/L WAF

1

17.55

17.41

18.03

17.98

16.76

16.92

18.25

18.60

17.00

17.46

17.59

18.34

2

18.13

17.13

17.35

17.14

16.66

17.26

17.71

17.80

17.36

17.91

17.49

18.09

3

17.17

17.83

18.55

17.41

16.37

16.44

17.67

15.48

18.80

18.51

18.48

17.29

3.2 mg/L WAF

1

17.36

17.67

17.98

17.94

18.88

18.43

17.54

17.20

16.47

16.45

18.66

17.39

2

19.27

16.67

18.13

18.08

18.43

16.42

17.48

18.86

18.41

18.73

17.84

19.71

3

17.84

16.18

18.45

18.92

18.61

18.12

18.66

18.88

18.87

17.71

18.02

18.58

10 mg/L WAF

1

18.35

18.49

18.26

18.21

18.26

19.50

17.88

18.64

17.06

18.01

17.71

16.70

2

17.77

16.20

18.20

17.17

16.29

18.10

19.14

18.72

17.79

16.79

18.00

17.82

3

17.16

16.88

16.07

16.99

17.97

18.38

18.62

17.85

17.76

18.81

16.29

17.59

32 mg/L WAF

1

17.93

16.01

18.36

19.08

17.98

18.05

17.39

17.43

19.38

17.41

17.98

18.32

2

17.81

17.84

18.85

18.32

19.19

17.45

19.65

18.35

18.57

17.78

17.31

18.17

3

16.09

18.83

18.33

16.96

18.25

18.28

17.21

18.83

15.46

15.88

17.92

18.37

100 mg/L WAF

1

17.14

18.58

17.82

16.21

18.56

18.05

18.89

17.55

18.80

18.15

17.02

18.63

2

14.94

17.89

14.92

16.82

18.87

18.28

16.71

18.43

17.45

17.74

17.03

17.19

3

17.92

18.19

17.77

13.13

18.70

18.70

18.51

15.44

18.81

18.15

10.78

18.98

Note: “-“ not measured, because the larvae or juvenile fish were dead.

The total length of larvae or juvenile fish at the end of the test (continuation)

The nominal loading rate

Replicate

Total length (mm)

13

14

15

16

17

18

19

20

M

SD

CV(%)

Blank control

1

16.63

16.61

15.91

15.50

17.00

17.92

15.35

-

16.98

0.86

1.24

2

17.71

17.99

17.29

17.65

16.71

16.78

17.04

18.00

17.35

0.79

3

18.00

17.43

17.11

18.65

18.25

19.46

17.16

-

17.35

1.01

1.0 mg/L WAF

1

18.25

14.63

17.16

17.81

16.01

17.65

18.16

17.40

17.45

0.91

0.44

2

16.69

17.09

16.80

18.04

17.70

17.61

16.88

-

17.41

0.47

3

17.71

17.77

16.76

17.78

17.66

18.37

-

-

18.56

88

3.2 mg/L WAF

1

18.51

18.88

17.74

19.49

18.17

16.30

17.80

16.78

17.78

0.88

0.44

2

17.71

16.47

17.72

18.56

16.62

17.70

-

-

17.93

0.95

3

18.92

16.52

16.83

17.98

17.62

14.89

17.50

18.84

17.90

1.08

10 mg/L WAF

1

17.31

18.60

18.40

17.69

18.23

18.09

18.77

18.58

18.14

0.63

2.77

2

17.70

17.01

16.82

18.66

18.97

8.12

-

-

17.18

2.42

3

17.82

17.03

16.51

17.94

18.14

17.08

-

-

17.49

0.79

32 mg/L WAF

1

20.33

19.05

18.47

16.41

17.38

17.45

18.61

17.54

18.03

1.00

1.48

2

18.04

17.52

18.77

16.29

18.81

18.82

18.96

-

18.24

0.79

3

17.18

18.89

19.12

16.52

18.07

18.02

18.25

-

17.71

1.09

100 mg/L WAF

1

17.55

18.08

17.65

18.59

18.60

18.00

-

-

17.99

0.71

1.18

2

17.70

18.51

17.98

17.60

18.33

18.39

18.41

18.68

17.59

1.10

3

18.66

18.12

19.75

18.13

19.62

18.88

-

-

17.68

2.30

Note: “-“ not measured, because the larvae or juvenile fish were dead.

 

The dry weights of larvae or juvenile fish at the end of the test

The nominal loading rate

Replicate

Weight of dish (g)

Weight of dish + fish (g)

Number of fish (individual)

The weight of individual fish (mg)

Mean (mg)

SD (mg)

CV (%)

Blank control

1

85.546

85.747

19

10.58

10.96

0.34

3.08

2

86.246

86.468

20

11.10

3

90.106

90.319

19

11.21

1.0 mg/L WAF

1

85.836

86.070

20

11.70

11.40

0.28

2.43

2

86.061

86.273

19

11.16

3

76.891

77.095

18

11.33

3.2 mg/L WAF

1

88.120

88.362

20

12.10

11.78

0.30

2.56

2

69.198

69.405

18

11.50

3

81.253

81.488

20

11.75

10 mg/L WAF

1

84.204

84.453

20

12.45

11.65

0.91

7.77

2

76.081

76.273

18

10.67

3

85.891

86.104

18

11.83

32 mg/L WAF

1

85.914

86.157

20

12.15

12.59

0.85

6.79

2

82.584

82.842

19

13.58

3

90.363

90.592

19

12.05

100 mg/L WAF

1

87.692

87.914

18

12.33

12.06

0.67

5.55

2

94.553

94.779

20

11.30

3

75.383

75.609

18

12.56

 

 

The pH of all solutions during the test

The nominal loading rate

Replicate

0d

1d

7d

8d

10d

11d

14d

15d

21d

22d

27d

28d

33d

34d

New

Old

New

Old

New

Old

New

Old

New

Old

New

Old

New

Old

Blank control

1

8.36

8.23

8.15

7.71

8.27

7.80

8.01

7.86

8.09

7.85

7.89

7.68

8.07

7.66

2

8.36

8.26

8.19

7.74

8.25

7.81

8.00

7.89

8.09

7.85

7.86

7.65

7.95

7.65

3

8.36

8.28

8.12

7.83

8.29

7.70

7.91

7.90

8.15

7.85

7.87

7.65

7.95

7.64

1.0 mg/L WAF

1

8.34

8.25

8.16

7.65

8.24

7.80

8.17

7.99

8.10

7.85

7.86

7.55

7.95

7.68

2

8.34

8.26

8.18

7.70

8.24

7.71

8.18

7.96

8.1

7.87

7.84

7.55

7.99

7.69

3

8.34

8.26

8.15

7.60

8.23

7.72

8.19

7.93

8.12

7.87

7.89

7.55

7.97

7.70

3.2 mg/L WAF

1

8.35

8.27

8.16

7.58

8.26

7.80

8.18

7.98

8.08

7.81

7.95

7.56

7.94

7.63

2

8.35

8.27

8.11

7.62

8.28

7.82

8.17

7.95

8.13

7.85

7.90

7.51

7.95

7.65

3

8.35

8.27

8.11

7.62

8.28

7.90

8.17

7.94

8.11

7.85

7.87

7.54

7.95

7.65

10 mg/L WAF

1

8.35

8.28

8.18

7.66

8.28

7.92

8.17

7.93

8.11

7.77

7.86

7.56

7.92

7.68

2

8.35

8.29

8.16

7.64

8.28

7.94

8.18

7.96

8.12

7.78

7.89

7.51

7.98

7.70

3

8.35

8.29

8.11

7.61

8.27

7.93

8.17

7.90

8.09

7.70

7.87

7.53

7.96

7.69

32 mg/L WAF

1

8.35

8.28

8.22

7.67

8.29

7.77

8.20

7.93

8.13

7.77

7.82

7.54

7.96

7.69

2

8.35

8.29

8.23

7.65

8.28

7.84

8.20

7.99

8.05

7.87

7.85

7.52

7.92

7.70

3

8.35

8.28

8.18

7.57

8.30

7.84

8.20

7.94

8.13

7.91

7.80

7.52

7.95

7.65

100 mg/L WAF

1

8.40

8.29

8.13

7.62

8.30

7.70

8.15

7.96

8.14

7.88

7.87

7.54

7.96

7.69

2

8.40

8.28

8.04

7.65

8.28

7.69

8.14

7.94

8.14

7.92

7.87

7.52

7.97

7.69

3

8.40

8.29

8.03

7.67

8.28

7.74

8.14

7.94

8.14

7.94

7.87

7.52

7.97

7.61

 

The dissolved oxygen concentration of test solution (%, air saturation value)

The nominal loading rate

Replicate

0d

1d

7d

8d

10d

11d

14d

15d

21d

22d

27d

28d

33d

34d

New

Old

New

Old

New

Old

New

Old

New

Old

New

Old

New

Old

Blank control

1

93

98

85

75

91

70

92

85

95

71

92

66

76

51

2

93

99

87

72

90

64

92

86

94

71

89

65

76

62

3

93

100

84

77

90

62

96

87

94

70

91

67

75

61

1.0 mg/L WAF

1

98

98

91

64

95

78

98

87

97

71

89

61

76

63

2

98

98

91

64

95

78

98

87

97

71

89

61

76

63

3

98

98

90

63

92

65

99

85

90

69

89

61

77

64

3.2 mg/L WAF

1

99

99

90

69

94

80

98

88

94

69

89

60

76

66

2

99

98

87

68

95

75

99

88

94

71

90

61

76

63

3

99

97

87

66

96

79

99

86

94

74

89

61

77

63

10 mg/L WAF

1

98

99

91

78

95

78

99

87

92

66

88

63

76

65

2

98

99

90

68

96

79

98

89

92

67

89

62

80

66

3

98

99

88

62

97

74

99

84

98

65

88

66

79

66

32 mg/L WAF

1

99

99

93

62

95

64

99

86

98

71

87

61

78

65

2

99

99

93

61

96

78

99

88

94

75

88

63

75

66

3

99

98

92

61

95

74

99

86

91

75

86

63

77

63

100 mg/L WAF

1

95

98

90

64

95

60

98

88

91

74

89

67

78

65

2

95

98

84

66

94

62

98

86

91

76

88

64

78

66

3

95

98

83

65

95

68

98

88

91

77

88

67

78

63

 

The temperature of test solutions measured regularly (°C)

The nominal loading rate

Replicate

0d

1d

7d

8d

10d

11d

14d

15d

21d

22d

27d

28d

33d

34d

New

Old

New

Old

New

Old

New

Old

New

Old

New

Old

New

Old

Blank control

1

24.9

25.5

24.7

24.3

25.2

25.4

24.5

24.5

24.6

24.1

24.8

24.8

25.2

24.9

2

24.9

25.5

24.7

24.3

25.2

25.4

24.5

24.5

24.6

24.1

24.8

24.8

25.2

24.9

3

24.9

25.5

24.7

24.3

25.2

25.4

24.5

24.5

24.6

24.1

24.8

24.8

25.2

24.9

1.0 mg/L WAF

1

24.9

25.5

24.7

24.3

25.2

25.4

24.5

24.5

24.6

24.1

24.8

24.8

25.2

24.9

2

24.9

25.5

24.7

24.3

25.2

25.4

24.5

24.5

24.6

24.1

24.8

24.8

25.2

24.9

3

24.9

25.5

24.7

24.3

25.2

25.4

24.5

24.5

24.6

24.1

24.8

24.8

25.2

24.9

3.2 mg/L WAF

1

24.9

25.5

24.7

24.3

25.2

25.4

24.5

24.5

24.6

24.1

24.8

24.8

25.2

24.9

2

24.9

25.5

24.7

24.3

25.2

25.4

24.5

24.5

24.6

24.1

24.8

24.8

25.2

24.9

3

24.9

25.5

24.7

24.3

25.2

25.4

24.5

24.5

24.6

24.1

24.8

24.8

25.2

24.9

10 mg/L WAF

1

24.9

25.5

24.7

24.3

25.2

25.4

24.5

24.5

24.6

24.1

24.8

24.8

25.2

24.9

2

24.9

25.5

24.7

24.3

25.2

25.4

24.5

24.5

24.6

24.1

24.8

24.8

25.2

24.9

3

24.9

25.5

24.7

24.3

25.2

25.4

24.5

24.5

24.6

24.1

24.8

24.8

25.2

24.9

32 mg/L WAF

1

24.9

25.5

24.7

24.3

25.2

25.4

24.5

24.5

24.6

24.1

24.8

24.8

25.2

24.9

2

24.9

25.5

24.7

24.3

25.2

25.4

24.5

24.5

24.6

24.1

24.8

24.8

25.2

24.9

3

24.9

25.5

24.7

24.3

25.2

25.4

24.5

24.5

24.6

24.1

24.8

24.8

25.2

24.9

100 mg/L WAF

1

24.9

25.5

24.7

24.3

25.2

25.4

24.5

24.5

24.6

24.1

24.8

24.8

25.2

24.9

2

24.9

25.5

24.7

24.3

25.2

25.4

24.5

24.5

24.6

24.1

24.8

24.8

25.2

24.9

3

24.9

25.5

24.7

24.3

25.2

25.4

24.5

24.5

24.6

24.1

24.8

24.8

25.2

24.9

 

The results of salinity measured regularly (%)

The nominal loading rate

Replicate

0d

1d

7d

8d

10d

11d

14d

15d

21d

22d

27d

28d

33d

34d

New

Old

New

Old

New

Old

New

Old

New

Old

New

Old

New

Old

Blank control

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1.0 mg/L WAF

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3.2 mg/L WAF

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

10 mg/L WAF

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

32 mg/L WAF

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

100 mg/L WAF

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

The measured concentration of test solutions

 

 

The measured concentration (mg/L)

The nominal loading rate

Replicate

0d

1d

7d

8d

10d

11d

14d

15d

21d

22d

27d

28d

33d

34d

New

Old

New

Old

New

Old

New

Old

New

Old

New

Old

New

Old

Blank control

1

<LOD

<LOD

-

-

-

-

-

-

-

-

-

-

-

-

2

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

3

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

1.0 mg/L WAF

1

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

2

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

3

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

3.2 mg/L WAF

1

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

2

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

3

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

10 mg/L WAF

1

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

2

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

3

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

32 mg/L WAF

1

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

2

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

3

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

100 mg/L WAF

1

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

2

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

3

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

Note: Limit of detection (LOD) and limit of quantification (LOQ) of test substance were 0.645 mg/L and 2.15 mg/L, respectively, “<LOD” means lower than the LOD, “-“ means not detected.

Validity criteria fulfilled:
yes
Conclusions:
The test substance is a kind of UVCB item, and insoluble in test water, so water accommodate fraction was used to prepare test solution and the test results were expressed as the nominal loading rate. Under this test condition, at the end of the test (34 d), the NOEL and LOEL of Fish, Early-life Stage Toxicity Test to Rare Minnow (Gobiocypris rarus) are shown as follow:
Hatching rate: NOEL =100mg/L; LOEL >100mg/L
Surviving rate after post-hatched: NOEL =100mg/L; LOEL >100mg/L
Cumulative abnormal rate: NOEL =100mg/L; LOEL >100mg/L
Cumulative mortality: NOEL =100mg/L; LOEL >100mg/L
Total length of surviving fish: NOEL =100mg/L; LOEL >100mg/L
Dry weight of surviving fish: NOEL =100mg/L; LOEL >100mg/L
Executive summary:

Purpose of the test

Tests with the early-life stages of fish are intended to define the lethal and sub-lethal effects of chemicals on the stages and species tested. They yield information of value for the estimation of the chronic lethal and sub-lethal effects of the chemical on other species.

 

Guidelines

[1] State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, 210 Fish, Early-life Stage Toxicity Test [M]. Second Edition. Beijing: China Environmental Science Press, 2013:86-93.

[2] China National Technical Committee for Standardization of Dangerous Chemical Management GB/T 21854-2008.Chemical Fish, Early-life Stage Toxicity Test [S]. Beijing: China Standardization Press, 2008.

[3]OECD. OECD Guidelines for Testing of Chemicals, 210 Fish, early-life stage toxicity test. Paris: OECD, 26 July 2013.

[4] OECD. OECD series on testing and assessment number 23 (Second Edition), Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals. Paris: OECD, Adopted on February 8, 2019.

[5] ECETOC. Aquatic Toxicity Testing of Sparingly Soluble, Volatile and Unstable Substances [S]. ECETOC Monograph No. 26 1996.

[6] ASTM international. Standard Practice for Aquatic Toxicity Testing of Lubricants: Sample Preparation and Results Interpretation. D6081 − 98 (Reapproved 2014).

[7] State Environmental Protection Administration of China. HJ/T 153-2004, The Guidelines for the test of chemical [S]. Beijing: China Environmental Science Press.2004.

 

Principle

The early-life stages of fish are exposed to a range of concentrations of the test solutions. The test method of semi-static conditions are acceptable in this test. The test is initiated by placing fertilized eggs in test chambers and is continued for a species-specific time period that is necessary for the control fish to reach a juvenile life-stage. Lethal and sub-lethal effects are assessed and compared with control values to determine the lowest observed effect loading rate (LOEL) in order to determine the no observed effect loading rate (NOEL) and/or ELx (e.g. EL10, EL20) by using a regression model to estimate the concentration that would cause a x % change in the effect measured.

 

Test conditions

(1) Test substance: SynNova Base Oil

(2) Test organism: Embryos of Rare Minnow (within 8 hours after fertilisation)

(3) Exposure duration: 34 d

(4) Test concentration: According to the results of preliminary test (see Annex C), five nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L water accommodated fraction (WAF) test groups with a geometric factor of 3.2 and one blank control were set in the definitive test.

(5) Replicate: 3 replicates / test group

(6) Number of embryo: 20 embryos/ replicate, 60 embryos / test group, 360 embryos in total.

(7) Test water: Dechlorinated tap water

(8) Type of test: Renewal more than 3/4 volume test solution every 24 h by the way of semi-static test.

(9) Preparation of the test solution : 1d prior to the test – 8 d: 5.0 mg, 16 mg, 50 mg, 160 mg and 500 mg test substance were weighed into five flasks and mixed with small amount of dechlorinated tap water, then the mixtures were transferred intovessels and diluted with dechlorinated tap water to 5000 mL respectively to obtain the nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L. The solutions were stirred for about 24 h (the depth of vortex was maintained at one third of the test solution height) and stood for about 1 h, then 2000 mL of middle layer solutions were collected into anothervessels (the first 100 – 200 mL solution was discarded), the nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF solutions were obtained and divided into three replicates as test solutions ( 200 mL / replicate).

9 d – 32 d: 10 mg, 32 mg, 100 mg, 320 mg and 1000 mg test substance were weighed into five flasks and mixed with small amount of dechlorinated tap water, then the mixtures were transferred intovessels and diluted with dechlorinated tap water to 10000 Ml respectively to obtain the nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L suspensions. The suspensions were stirred for about 24 h (the depth of vortex was maintained at one third of the test solution height) and stood for about 1 h, then 7000 mL of middle layer solutions were collected into anothervessels (the first 100 – 200 mL solution was discarded), the nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF solutions were obtained and divided into three replicates as test solutions (2000 mL / replicate). The above mentioned about preparation of test solution is in accordance with the OECD guidance document No. 23 (2019) and ASTM D6081 (2014).

(10) Volume of test solution: 0 d – 9 d: about 200 mL / test vessel, 10 d- 34 d: about 2000 mL/ test vessel.

(11) Temperature of test solutions: The temperature of all test vessels measured regularly ranged from 24.1 °C to 25.5 °C, measured continuously ranged from 24.0 °C to.

(12) Photoperiod: 14 h light / 10 h darkness

(13) Analysis of actual concentrations of test substance in test solutions: The actual concentrations of test substance in 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were measured at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d, 33 d (new solution) and 1 d, 8 d, 11 d, 15 d, 22 d, 28 d, 34 d (old solution) by Gas Chromatograph (GC); the blank control was measured at 0 d (new solution) and 1 d (old solution).

 

Results

(1) Basis of preparation test solution

The test substance SynNova Base Oil is liquid - water white colorless oil. The test substance is a complex mixture / UVCB substance (Substances of Unknown or Variable Composition, Complex Reaction Products or Biological Materials) and is poorly soluble in test water. The aqueous media were prepared following ASTM D6081-98/Reapproved 2014), which is specifically developed for aquatic toxicity testing of lubricants, by mixing the test substance with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the WAF was separated by siphon. The WAF was checked for any undissolved or emulsified material by microscopic inspection, which was negative. Exposure was expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate). This procedure was followed for each renewal of the test solutions.

(2) The measured concentrations of test substance in test solutions

The results of the analytical methods are shown in the final report of “Validation of an Analytical Method of SynNova Base Oil” (The report No. is A19058-T19054-1R). Limit of detection (LOD) and limit of quantification (LOQ) of analytical method were 0.645 mg/L and 2.15 mg/L, respectively.

The measured concentrations of test substance in the blank control at 0 d (new solution) and 1 d (old solution) were both lower than LOD of 0.645 mg/L.

The measured concentrations of test substance in nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d, 33 d (new solution) and 1 d, 8 d, 11 d, 15 d, 22 d, 28 d, 34 d (old solution) were all lower than LOD of 0.645 mg/L. The measured concentrations of all test solutions lower than the LOD, this did not mean that there was no test substance present in the test solutions, just that the concentration was below the LOD of the analytical method, indicating that test substance was poorly soluble in test water.

(3) Toxicity effects on test organism

The test substance is a kind of UVCB item, and insoluble in test water, so water accommodate fraction was used to prepare test solution and the test results were expressed as the nominal loading rate. Under this test condition, at the end of the test (34 d), the NOEL and LOEL of Fish, Early-life Stage Toxicity Test to Rare Minnow (Gobiocypris rarus) are shown as follow:

Hatching rate: NOEL =100mg/L; LOEL >100mg/L

Surviving rate after post-hatched: NOEL =100mg/L; LOEL >100mg/L

Cumulative abnormal rate: NOEL =100mg/L; LOEL >100mg/L

Cumulative mortality: NOEL =100mg/L; LOEL >100mg/L

Total length of surviving fish: NOEL =100mg/L; LOEL >100mg/L

Dry weight of surviving fish: NOEL =100mg/L; LOEL >100mg/L

Endpoint:
fish short-term toxicity test on embryo and sac-fry stages
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 5, 2019 to November 15, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 212 (Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages)
Version / remarks:
OECD Guidelines for Testing of Chemicals, 212 Fish, Short-term toxicity Test on Embryo and Sac-fry Stage. Paris: OECD, Adopted 21 September 1998.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: National Guideline
Version / remarks:
State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, 212 Fish, short-term Toxicity Test on Embryo and sac-fry stages. Second Edition. Beijing: China Environmental Science Press, 2013:113-123.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: National Guideline
Version / remarks:
China National Technical Committee for Standardization of Dangerous Chemical Management GB/T 21807-2008. Chemical Fish Short-term test toxicity on Embryo and Sac-fry Stages [S]. Beijing: China Standardization Press, 2008.
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
No further details specified in the study report.
Analytical monitoring:
yes
Details on sampling:
The actual concentrations of test substance in test solution of 100 mg/L WAF test group were measured at 0 d, 3 d, 7 d (new solution) and 1 d, 4 d, 8 d (old solution). Test solution of blank control was measured at 0 d and 1 d (old solution).
Vehicle:
no
Details on test solutions:
The test substance is a kind of UVCB item, and insoluble in test water, according to “Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals” (OECD number 23 Second Edition, February 8, 2019) and “Standard Practice for Aquatic Toxicity Testing of Lubricants: Sample Preparation and Results Interpretation”(ASTM international, D6081 − 98, Reapproved 2014)”, water accommodate fraction was prepared as test solution. The preparation method is shown as below:
200 mg test substance was weighed into a flask and mixed with small amount of dechlorinated tap water, the mixture was transferred into a 2 L beaker and diluted with dechlorinated tap water to 2000 mL to obtain the nominal loading rate of 100 mg/L. The solution was stirred for about 24 h (the depth of vortex was maintained at one third of the test solution height) and stood for about 1 h, then 600 mL of middle layer solution was siphoned into another beaker (the first 50 mL solution was discarded), the nominal loading rate of 100 mg/L WAF solution was obtained and divided into three replicates as test solution directly.
Test organisms (species):
other: Gobiocypris rarus
Details on test organisms:
(1) Species: Rare Minnow(Gobiocypris rarus)
(2) Reason for selection of species: The test species is recommended by “The Guidelines for the Testing of Chemicals, 212 Fish, Short-term Toxicity Test on Embryo and Sac-fry Stages” (Chemical Registration Center of MEP, Second Edition).
(3) Supplier: The brood fish with batch number FGr20181031-1 were provide by Safety Evaluation Center of Shenyang Chemical Research Institute CO. LTD.
(4) The standard of embryo: The embryos were not older than 8 h after fertilized
(5) Obtained embryo: The brood fish were hold in the water maintained the temperature at 25 °C ± 2 °C with 14 h light /10 h darkness. Before the test, the female and male was paired as 1:1, the embryos collected by artificial fertilisation.
(6) Selection and treatment of embryo: Embryos with normal development and good morphology were selected under an anatomical microscope. The normal division embryos are sticky and transparent. However, unfertilized embryos are observed as milky white.
(7) Number of embryo: 60 embryos
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
9 d
Post exposure observation period:
No post exposure observations period specified in the study report.
Hardness:
Total hardness of bank control and 100 mg/L WAF test group were measured at the beginning of the test
Test temperature:
he temperature of all test vessels measured at regularly ranged from 25.2 °C to 25.8 °C, and measured continuously ranged from 25.4 °C to 26.0 °C.
pH:
The test was performed without adjustment of pH and the pH was in the range of 8.21 to 8.36 during the test period.
Dissolved oxygen:
The dissolved oxygen concentration in the test solutions was kept at least 60% of the air saturation value throughout the test (ASV).
Salinity:
Salinity of bank control and 100 mg/L WAF test group were measured at the beginning of the test.
Conductivity:
No specified
Nominal and measured concentrations:
The measured concentrations of test substance in 100 mg/L WAF test group at 0 d, 3 d, 7 d (new solution) and 1 d, 4 d, 8 d (old solution) were all lower than LOD of 0.645 mg/L.
Details on test conditions:
Test water
Dechlorinated tap water was used in the test. Before the test, the test water was dechlorinated by aeration so that the dissolved oxygen concentration reached more than 80% of the air saturation value.

Design of the limit test
According to the results of preliminary test (see Annex C), cumulative abnormal rates of blank control, 10 mg/L WAF and 100 mg/L WAF test groups were all 0%. No abnormal appearances and behaviors were observed during the test. The measured concentrations of test substance were all lower than the limit of detection, so the stability of test substance could not be confirmed. Therefore, the limit test was performed under semi-static test at 24 h intervals with renewal more than 3/4 volume of test solution. One blank control and one nominal loading rate of 100 mg/L water accommodated fraction (WAF) test group were selected in the limit test, with 10 embryos in each replicate and 3 replicates for each test group.

Test conditions
Conditions of exposure
(1) Type of the test: The limit test was conducted as semi-static test with renew at least 3/4 volume test solution every 24 h (As the embryos were obtained at night of 0 d, in order to perform the test expediently, the time of the first renewal solution was changed on about 16 h after the start of the exposure).
(2) Exposure duration: 9 d (about 206 h)
(3) Test concentration: One nominal loading rate of 100 mg/L WAF group was selected in the test.
(4) Control: A blank control (test water without test substance) was set.
(5) Replicate: 3 replicates/test group
(6) Numbers of embryos: 10 embryos/test vessel, 30 embryos/test group, 60 embryos in total
(7) Volume of test solution: 200mL/test vessel

Conditions of test environment
(1) Water temperature: The temperature of all test vessels measured at regularly ranged from 25.2 °C to 25.8 °C, and measured continuously ranged from 25.4 °C to 26.0 °C.
(2) Dissolved oxygen concentration: The dissolved oxygen concentration in the test solutions was kept at least 60% of the air saturation value throughout the test (ASV).
(3) pH: The test was performed without adjustment of pH and the pH was in the range of 8.21 to 8.36 during the test period.
(4) Light: Artificial light of white fluorescent lamp was used. The photoperiod was 14 h light / 10 h darkness.

Observation and measurement
(1) Observation embryos and larvae
Stage of embryonic development: The embryonic stages were recorded at the beginning of exposure time.
Hatching and survival: Observations on hatching and survival were made daily and numbers recorded, removed dead individuals.
Abnormal appearance: The numbers of embryos or larvae showing abnormality of body form were recorded daily.
Abnormal behaviour: The behavour of larvae after hatching were observed and recorded daily.
(2) Temperature, pH and Dissolved oxidation: The temperature, pH and dissolved oxygen of all test vessels at 0 d, 3 d, 7 d (new solution) and 1 d, 4 d, 8 d (old solution) were measured. A test vessel with test water was put in the incubator and monitored continuously by a thermometer during the test.
(3) Total hardness of test solution: Total hardness of bank control and 100 mg/L WAF test group were measured at the beginning of the test.
(4) Salinity of test solution: Salinity of bank control and 100 mg/L WAF test group were measured at the beginning of the test.
(5) Measurement of individual lengths: Total lengths of surviving larvae were measured at the end of the test.
(6) Measurement the dry weights of larvae: The dry weights of survival larvae of each vessel were measured at the end of the test.
(7) Measured concentrations of test substance in test solutions: The actual concentrations of test substance in test solution of 100 mg/L WAF test group were measured at 0 d, 3 d, 7 d (new solution) and 1 d, 4 d, 8 d (old solution) by GC. Test solution of blank control was measured at 0 d and 1 d (old solution).

Data process
Cumulative mortality, numbers of healthy larvae, numbers of hatching larvae per day, lengths and weights of surviving larvae, numbers of larvae that are deformed or of abnormal appearance, numbers of larvae exhibiting abnormal behaviours were statistic. Alternatively, the normal distribution, variance of homogeneity, Independent T test were performed on the total length and dry weight of the surviving larvae with SPSS 20.0, to estimate the LOEL and NOEL by significant analysis between the blank control and test group. The Chi-Square Test was performed on the mortality rate and abnormality rate to estimate the NOEL and LOEL.
Reference substance (positive control):
not required
Key result
Duration:
9 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
9 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
number hatched
Key result
Duration:
9 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
9 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Key result
Duration:
9 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
9 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
number hatched
Key result
Duration:
9 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
9 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Details on results:
Observation of test solutions
The WAF solution was prepared with the direct addition method. During the preparation periods, surface films in the top layers were observed in all test solutions, so the solution in the middle layer was siphoned into another test vessel as exposure solution, certainly, there no obvious droplets or surface films were observed in the exposure solution. Any remaining non-dissolved test chemical component in the WAF has the possibility to cause physical effects on the test organism unrelated to inherent chemical toxicity.

Hatching rate, surviving rate after post-hatched, cumulative mortality and cumulative abnormal rate of embryos / larvae
The blastocyst stage of embryos that not older than 8 h after fertilized were exposed. All embryos were completely hatched at 4th day after start of the test, so the hatching rates of embryos in blank control and 100 mg/L WAF test groups were both 100%. The hatching rate of blank control had met the validity criterion that the hatched success should greater than 80%.
At the end of test, the surviving rate after post-hatched of the blank control was 100%, which met the validity criterion that the surviving rate after post –hatched should not less than 90%. The surviving rate of 100 mg/L WAF test groups was 100%. So based on the surviving rate after post-hatched, the NOEL was 100 mg/L WAF, and the LOEL was greater than 100 mg/L WAF.
At the end of test, the cumulative mortalities of embryos / larvae in blank control and 100 mg/L WAF test groups were both 0%. So based on the cumulatively mortality, the NOEL was 100 mg/L and the LOEL was greater than 100mg/L.
At the end of test, the cumulative abnormal rates of embryos / larvae in blank control and 100 mg/L WAF test groups were both 0%. So based on the cumulatively abnormal rate, the NOEL was 100 mg/L and the LOEL was greater than 100 mg/L.

The abnormal appearances of embryos and larvae
During the test, no abnormal appearances were observed in blank control and 100 mg/L WAF test group.

The abnormal behaviors of larvae
During the test, No abnormal behaviors were observed in the blank control and 100 mg/L WAF test group.

The total lengths of surviving larvae
At the end of the test, the mean and the standard deviation of total length of surviving larvae in blank control was 5.41 mm ± 0.22 mm, coefficient of variation (CV) was 4%, which was met the validity criterion that the CV should not exceed 20%.
The mean and standard deviation of total length in 100 mg/L WAF test group was 5.42 mm ± 0.14 mm.

The dry weights of surviving larvae
At the end of the test, the average and standard deviation of dry weight of surviving larvae in blank control was 0.08 mg± 0.02 mg, CV was 19%, which was met the validity criterion that the CV should not exceed 20%.
The mean and standard deviation of dry weight in 100 mg/L WAF test group was 0.11 mg ± 0.02 mg. So based on the dry weight of surviving larvae, the NOEL was the loading rate of 100 mg/L and the LOEL was greater than loading rate of 100 mg/L.

The temperature, pH, dissolved oxygen, hardness and salinity
During the test, the pH of the test solution was ranged from 8.21 to 8.36. The temperature of all test vessels measured regularly ranged from 25.2 °C to 25.8 °C, and measured continuously ranged from 25.4 °C to 26.0 °C, the temperature was not differ by more than ± 1.5 °C between test chambers or between successive days at any time during the test, and the temperature was within the range of 25°C +/- 1°C. The dissolved oxygen concentration ranged from 92% to 101% of the air saturation value (ASV) and met the requirement of at least 60% of ASV.
At the beginning of the test, the total hardness of the blank control and 100 mg/L WAF test substance group were both 110 mg/L, and the salinity were both 0‰.

Analytical concentration of test substance in test solution
Sample pretreatment and preservation
The test solutions were collected and filtered through 0.45 μm aqueous phase PES filter membranes (the first 5 mL - 10 mL filtrate was discarded), 50 mL filtrate was transferred into a separating funnel and extract three times with 20 mL, 15 mL and 10 mL of n-hexane, respectively. The three extracts were collected into a 50 mL volume flask and diluted to 50 mL with n-hexane before analyzed. The samples were analyzed immediately, otherwise the samples were stored at 2 °C – 8 °C and protected from light after pretreatment, and the measurement were carried out within 96 h.

Measured concentrations of test substance
Limit of detection (LOD) and limit of quantification (LOQ) of analytical method were 0.645 mg/L and 2.15 mg/L, respectively.
The measured concentrations of test substance in the blank control at 0 d (new solution) and 1 d (old solution) were both lower than LOD of 0.645 mg/L.
The measured concentrations of test substance in 100 mg/L WAF test group at 0 d, 3 d, 7 d (new solution) and 1 d, 4 d, 8 d (old solution) were all lower than LOD of 0.645 mg/L. The measured concentrations of all test solutions lower than the LOD, this did not mean that there was no test substance present in the test solutions, just that the concentration was below the LOD of the analytical method, indicating that test substance was poorly soluble in test water.

Validity of test
(1) The hatching rate of blank control was 100%, and the surviving rate after post-hatched was 100%, which met the validity criterion that the hatching rate should not less than 80% and the surviving rate after post-hatched should not less than 90%.
(2) The dissolved oxygen of test solution was ranged from 92% to 101% of ASV, which met the validity criterion that dissolved oxygen must have been at least 60% of ASV.
(3) The temperature of all test vessels measured regularly ranged from 25.2 °C to 25.8 °C, and measured continuously ranged from 25.4 °C to 26.0 °C, the temperature was not differ by more than ±1.5 °C between test chambers or between successive days at any time during the test, and the temperature was within the range of 25°C +/- 1°C.
Results with reference substance (positive control):
Not required
Reported statistics and error estimates:
The Mann-Whitney U test was performed, the results showed that based on the total length of surviving larvae, there was no statistically significant difference (p>0.05) between the blank control and the limit loading rate of 100 mg/L, so the NOEL was the loading rate of 100 mg/L, and the LOEL was greater than the loading rate of 100 mg/L .
Results of Independent T test showed that, there was no statistically significant difference (p>0.05) based on dry weight between the blank control and 100 mg/L WAF test group.

Hatching rate, surviving rate after post-hatched, cumulative mortality and cumulative abnormal rate of embryos/larvae at the end of test

Nominal loading rate

Replicate

Total embryos

Number of dead embryos

Mortality of embryos

Number of abnormal embryos

Abnormal rate of embryos

Hatching rate

Total larvae

Number of dead larvae

Mortality of larvae

Surviving rate of larvae

Number of abnormal larvae

Abnormal rate of larvae

Cumulative mortality

Cumulative abnormal rate

Blank control

1

10

0

0%

0

0%

100%

10

0

0%

100%

0

0%

0%

0%

2

10

0

0%

0

0%

10

0

0%

0

0%

3

10

0

0%

0

0%

10

0

0%

0

0%

100 mg/L WAF

1

10

0

0%

0

0%

100%

10

0

0%

100%

0

0%

0%

0%

2

10

0

0%

0

0%

10

0

0%

0

0%

3

10

0

0%

0

0%

10

0

0%

0

0%

 

The abnormal appearances of embryos and larvae during the test

Nominal loading rate

Replicate

Result (left: number of abnormal embryo or larvae /number of survival embryo or larvae; right: type of appearance (number))

1d

2d

3d

4d

5d

6d

7d

8d

9d

Blank control

1

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

2

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

3

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

100 mg/L WAF

1

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

2

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

3

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

Note: “N” means normal.

 

The abnormal behaviours of larvae during the test

Nominal loading rate

Replicate

Result (left: number of abnormal larvae/number of hatched and survival larvae; right: type of behaviour)

4d

5d

6d

7d

8d

9d

Blank control

1

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

2

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

3

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

100 mg/L WAF

1

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

2

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

3

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

N

Note: “N” means normal. The abnormal behaviours were observed after post-hatched.

 

The body length of survival larvae at the end of test

Nominal loading rate

Replicate

Body length of larvae (mm)

Average (mm)

Total average (mm)

Standard deviation (mm)

Total standard deviation (mm)

Coefficient of variation

1

2

3

4

5

6

7

8

9

10

Blank control

1

5.25

5.10

4.90

5.14

6.06

5.46

5.55

5.44

5.38

5.24

5.35

5.41

0.32

0.22

4%

2

5.63

5.44

5.47

5.56

5.37

5.53

5.47

5.18

5.58

5.28

5.45

0.15

3

5.17

5.65

5.49

5.57

5.53

5.49

5.26

5.45

5.33

5.26

5.42

 

100 mg/L WAF

1

5.49

5.58

5.13

5.61

5.55

5.30

5.24

5.52

5.38

5.31

5.41

5.42

0.17

0.14

2%

2

5.71

5.42

5.44

5.47

5.45

5.54

5.13

5.52

5.40

5.40

5.45

0.15

3

5.44

5.50

5.26

5.41

5.49

5.52

5.46

5.41

5.22

5.34

5.41

0.11

 

The dry weight of survival larvae at the end of test

Nominal loading rate

Replicate

Dry weight of dish (g)

Dry weight of dish + larvae (g)

Dry weight of larvae (g)

Number of larvae

Average of dry weight of individual larvae (g)

Average (g)

Standard deviation (g)

Coefficient of variation

Blank control

1

45413.10

45414.10

1.00

10

0.1000

0.083

0.016

19%

2

40524.79

40525.49

0.700

10

0.0700

3

40518.34

40519.12

0.780

10

0.0780

100 mg/L WAF

`

1

42334.00

42334.89

0.890

10

0.0890

0.109

0.018

16%

2

47304.83

47306.03

1.200

10

0.1200

3

42332.23

42333.42

1.190

10

0.1190

 

The pH of test solution during the test

Nominal loading rate

Replicate

0d

1d

3d

4d

7d

8d

New

Old

New

Old

New

Old

Blank control

1

8.36

8.33

8.30

8.27

8.33

8.21

2

8.36

8.33

8.32

8.26

8.34

8.25

3

8.36

8.33

8.31

8.27

8.36

8.24

100 mg/L WAF

1

8.36

8.32

8.30

8.28

8.33

8.29

2

8.36

8.33

8.29

8.28

8.34

8.27

3

8.36

8.34

8.30

8.27

8.34

8.27

 

The dissolved oxygen of test solution during the test

Nominal loading rate

Replicate

0d

1d

3d

4d

7d

8d

New

Old

New

Old

New

Old

Blank control

1

93

100

96

95

92

100

2

93

99

97

94

93

97

3

93

100

98

95

93

98

100 mg/L WAF

1

98

100

99

96

96

97

2

98

99

101

98

97

96

3

98

98

98

95

97

97

 

The temperature of all test vessels measured regularly (°C)

Nominal loading rate

Replicate

0d

1d

3d

4d

7d

8d

New

Old

New

Old

New

Old

Blank control

1

25.2

25.5

25.7

25.4

25.6

25.8

2

25.2

25.5

25.7

25.4

25.6

25.8

3

25.2

25.5

25.7

25.4

25.6

25.8

100 mg/L WAF

1

25.2

25.5

25.7

25.4

25.6

25.8

2

25.2

25.5

25.7

25.4

25.6

25.8

3

25.2

25.5

25.7

25.4

25.6

25.8

 

Measured concentrations of test substance in test solution

Nominal loading rate

Measured concentration of test substance (mg/L)

0d

New

1d

Old

3d
New

4d

Old

7d

New

8d

Old

Blank control

<LOD

<LOD

-

-

-

-

100 mg/L WAF

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

Note: Limit of detection (LOD) of test substance was 0.645 mg/L. “<LOD” means lower that the LOD.

Validity criteria fulfilled:
yes
Conclusions:
The test substance is a kind of UVCB item, and insoluble in test water, so water accommodate fraction was used to prepare test solution and the test results were expressed as the nominal loading rate. Under this test condition, at the end of the test (9 d), the NOEL and LOEL to embryos of Rare Minnow (Gobiocypris rarus) are shown as follow:

Hatching rate: NOEL =100 mg/L; LOEL >100 mg/L
Surviving rate of larvae after post-hatched: NOEL =100 mg/L; LOEL >100 mg/L
Cumulative abnormal rate of embryos/larvae: NOEL =100 mg/L; LOEL >100 mg/L
Cumulative mortality of embryos/larvae: NOEL =100 mg/L; LOEL >100 mg/L
Total length of surviving larvae: NOEL =100 mg/L; LOEL >100 mg/L
Dry weight of surviving larvae: NOEL =100 mg/L; LOEL >100 mg/L
Executive summary:

Purpose of the test

The aimed of the study on fish, short-term toxicity test on embryo and sac-fry stages would be intend to define lethal and sub-lethal effects on the test substance.

 

Guidelines

 [1] State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, 212 Fish, short-term Toxicity Test on Embryo and sac-fry stages. Second Edition. Beijing: China Environmental Science Press, 2013:113-123.

[2] China National Technical Committee for Standardization of Dangerous Chemical Management GB/T 21807-2008. Chemical Fish Short-term test toxicity on Embryo and Sac-fry Stages [S]. Beijing: China Standardization Press, 2008.

[3] OECD. OECD Guidelines for Testing of Chemicals, 212 Fish, Short-term toxicity Test on Embryo and Sac-fry Stage. Paris: OECD, Adopted 21 September 1998.

[4] OECD. OECD series on testing and assessment number 23 (Second Edition), Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals. Paris: OECD, Adopted on February 8, 2019.

[5] ECETOC. Aquatic Toxicity Testing of Sparingly Soluble, Volatile and Unstable Substances [S]. ECETOC Monograph No. 26 1996.

[6] ASTM international. Standard Practice for Aquatic Toxicity Testing of Lubricants: Sample Preparation and Results Interpretation. D6081 − 98 (Reapproved 2014).

[7] State Environmental Protection Administration of China. HJ/T 153-2004, The Guidelines for the test of chemical [S]. Beijing: China Environmental Science Press.2004.

 

Principle

The embryo and sac-fry stages of fish are exposed to a range of concentrations of the test substance dissolved in water under semi-static conditions. The test is begun by placing fertilized eggs in the test chambers and is terminated just before the yolk-sac of any larvae in any of the test chambers has been completely absorbed or before mortalities by starvation start in controls. Lethal and sub-lethal effects are assessed and compared with control values to determine the lowest observed effect loading rate (LOEL) and hence the no observed effect loading rate (NOEL). Alternatively, they may be analyzed using a regression model in order to estimate the loading rate that would cause a given percentage effect (i.e. LLx / ELx where x is a defined % effect).

 

Test conditions

(1) Test substance: SynNova Base Oil

(2) Test organism: Embryos of Rare Minnow (within 8 hours after fertilisation)

(3) Exposure duration: 9 d (about 206 h)

(4) Test concentration: According to the results of preliminary test (see Annex C), the limit test was conducted with one blank control and one nominal loading rate of 100 mg/L water accommodated fraction (WAF) test group.

(5) Replicate: 3 replicates / test group

(6) Number of embryo: 10embryos/ replicate, 30 embryos / test group, 60 embryos in total.

(7) Test water: Dechlorinated tap water

(8) Type of test: Renewal more than 3/4 volume test solution every 24 h by the way of semi-static test.

(9) Preparation of the test solution: 200 mg test substance was weighed into a flask and mixed with small amount of dechlorinated tap water, the mixture was transferred into a 2L beaker and diluted with dechlorinated tap water to 2000 mL to obtain the nominal loading rate of 100 mg/L. The solution was stirred for about 24 h (the depth of vortex was maintained at one third of the test solution height) and stood for about 1 h, then 600 mL of middle layer solution was siphoned into another beaker (the first 50 mL solution was disgarded), the nominal loading rate of 100 mg/L WAF solution was obtained and divided into three replicates as test solution directly. This procedure is in accordance with the OECD guidance document No. 23 (2019) and ASTM D6081 (2014).

(10) Volume of test solution: about 200 mL / test vessel

(11) Temperature of test solutions: The temperature of all test vessels measured regularly ranged fromto, measured continuously ranged fromto.

(12) Photoperiod: 14 h light / 10 h darkness

(13) Analysis of actual concentrations of test substance in test solutions: The actual concentrations of test substance in blank control and 100 mg/L WAF test groups were measured at 0 d, 3 d, 7 d (new solution) and 1 d, 4 d, 8 d (old solution) by Gas Chromatography (GC). Test solution of blank control was measured at 0 d and 1 d (old solution).

 

Results

(1) Basis of preparation test solution

The test substance SynNova Base Oil is liquid - water white colorless oil. The test substance is a complex mixture / UVCB substance (Substances of Unknown or Variable Composition, Complex Reaction Products or Biological Materials) and is poorly soluble in test water. The aqueous media were prepared following ASTM D6081-98/Reapproved 2014), which is specifically developed for aquatic toxicity testing of lubricants, by mixing the test substance with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the WAF was separated by siphon. The WAF was checked for any undissolved or emulsified material by microscopic inspection, which was negative. Exposure was expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate). This procedure was followed for each renewal of the test solutions.

(2) The measured concentrations of test substance in test solutions

The results of the analytical methods are shown in the final report of “Validation of an Analytical Method of SynNova Base Oil” (The report No. is A19058-T19054-1R). Limit of detection (LOD) and limit of quantification (LOQ) of analytical method were 0.645 mg/L and 2.15 mg/L, respectively.

The measured concentrations of test substance in the blank control at 0 d (new solution) and 1 d (old solution) were both lower than LOD of 0.645 mg/L.

The measured concentrations of test substance in 100 mg/L WAF test group at 0 d, 3 d, 7 d (new solution) and 1 d, 4 d, 8 d (old solution) were all lower than LOD of 0.645 mg/L. The measured concentrations of all test solutions lower than the LOD, this did not mean that there was no test substance present in the test solutions, just that the concentration was below the LOD of the analytical method, indicating that test substance was poorly soluble in test water.

(3) Toxicity effects on test organism

The test substance is a kind of UVCB item, and insoluble in test water, so water accommodate fraction was used to prepare test solution and the test results were expressed as the nominal loading rate. Under this test condition, at the end of the test (9 d), the No Observed Effect loading rate (NOEL) and Lowest Observed Effect loading rate (LOEL) to embryos of Rare Minnow (Gobiocypris rarus) are shown as follow:

 

Hatching rate: NOEL =100 mg/L; LOEL >100 mg/L

Surviving rate of larvae after post-hatched: NOEL =100 mg/L; LOEL >100 mg/L

Cumulative abnormal rate of embryos/larvae: NOEL =100 mg/L; LOEL >100 mg/L

Cumulative mortality of embryos/larvae: NOEL =100 mg/L; LOEL >100 mg/L

Total length of surviving larvae: NOEL =100 mg/L; LOEL >100 mg/L

Dry weight of surviving larvae: NOEL =100 mg/L; LOEL >100 mg/L

Endpoint:
fish, juvenile growth test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 6, 2019 to December 6, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 215 (Fish, Juvenile Growth Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals, No. 215 Fish, Juvenile Growth Test[S]. OECD: Adopted 21 January, 2000.
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to guideline
Guideline:
other: National Guideline
Version / remarks:
State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, No. 215 Fish, Juvenile Growth Test. Second Edition. Beijing: China Environmental Science Press, 2013: 135-144.
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to guideline
Guideline:
other: National Guideline
Version / remarks:
China National Technical Committee for Standardization of Dangerous Chemical Management GBT 21806-2008. Chemical Fish, Juvenile Growth Test [S]. Beijing: China Standardization Press, 2008.
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes
Specific details on test material used for the study:
No further details specified in the study report.
Analytical monitoring:
yes
Details on sampling:
Test solutions of each test substance groups were collected at 0 d, 7 d, 14 d, 21 d (new solution) and 1 d, 8 d, 15 d, 22 d (old solution); test solution of blank control was collected at 0 d and 1 d (old solution), all the samples were used for analysis of test substance.
Vehicle:
no
Details on test solutions:
The test substance is a kind of UVCB item, and insoluble in test water, according to “Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals” (OECD number 23 Second Edition, February 8, 2019) and “Standard Practice for Aquatic Toxicity Testing of Lubricants: Sample Preparation and Results Interpretation” (ASTM international, D6081 − 98, Reapproved 2014)”, water accommodate fraction was used to prepare the test solution. The preparation method is shown as below:
5.0 mg, 16 mg, 50 mg, 160 mg and 500 mg test substance were weighed into flasks and mixed with small amount of dechlorinated tap water, then the mixtures were transferred into 5 L vessels and diluted with dechlorinated tap water to 5000 mL respectively, the nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L solutions were obtained. The solutions were stirred for about 24 h (the depth of vortex was maintained at one third of the test solution height) and stood for about 1 h, then 2000 mL of middle layer solutions were collected into another 5 L vessels (the first 100 – 200 mL solution was discarded), the nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF solutions were obtained and used as test solutions.
Note: After stirring, microscopic inspection was used to check whether the micro-droplet or insoluble test material presented in the test solution that collected at 0 d, 7 d, 14 d, 21 d were observed.
Test organisms (species):
other: Gobiocypris rarus
Details on test organisms:
Species: Rare Minnow (Gobiocypris rarus)
Batch number: FGr20190904-0906
Age: Approximately 62 d
Supplier: Breeding by the test laboratory
Size: The test fish were from a single population and the same batch.
Number of test fish: 60 in total (10 fish for each group)
Weight: The average weight of fish was 60 mg and the weights of the test fish ranged from 51 mg to 70 mg, all individual weight of fish was not exceed ± 25% of the average weight at the start of the test.
Reason for selection of species: The test species was recommended by 215 Fish Juvenile Growth Test from “The Guidelines for the Testing of Chemicals” published by the State Environmental Protection Administration of China.
Domestication: Before testing, those fish were domesticated for at least 14 d, the mortality rate was 0% during a 7 d acclimatization period. About 2% - 4% of the fish body weight per day were fed with artemia salina, and food was withheld from the fish for 24 hours prior to weight.

Food
Name: Artemia salina
Source: Artemia salina eggs were obtained from Shandong Beisen Feed Co., Ltd. The batch NO. was 20191031, and after hatched in the lab, the hatching brine shrimps were used as food.
Ingredient: Moisture 87.5%, dry matter content 12.5% (Crude protein 7.27%, crude fat 1.0%, ash content 1.9%)
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
28 d
Post exposure observation period:
No post exposure observation period specified in the study report.
Hardness:
Total hardness of blank control and 100 mg/L WAF test group were measured at the start of the test.
Test temperature:
The temperature of the test water:The temperature of each test vessel measured at regular intervals ranged from 22.2 ºC to 24.1 ºC, and the temperature detected continuously ranged from 22.2 ºC to 24.1 ºC during the test.
pH:
pH: 7.53 – 8.38.
Dissolved oxygen:
Dissolved oxygen: 63% - 100%, not less than 60% of air saturation value.
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
Test concentrations: five nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L.
Details on test conditions:
Test water
Dechlorinated tap water was used in the test. Before the test, the test water was dechlorinated by aeration so that the dissolved oxygen concentration reached more than 80% of the air saturation value.

Design of the definitive test
Results of the range finding test showed that, the measured concentration of test substance in test solutions were lower than the limit of detection, unable to determine whether the stability of test substance, consequently, the definitive test was performed under semi-static test at 24 h intervals. According to results of the preliminary tests, five nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L water accommodated fraction (WAF) test groups and one blank control were set in the definitive test. 10 fish for each test group and no replicates.

Preparation of the test system
Blank control: 2000 mL test water (dechlorinated tap water) was measured into 5 L test vessel as test solution of blank control.
Test substance groups: 2000 mL of WAF solutions were collected into 5 L test vessels as test solutions of each test substance groups.

Test conditions
Exposure conditions
(1) Test style: The semi-static test with renewal all test solution every 24 h
(2) Duration: 28 d
(3) Test concentrations: five nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L.
(4) Blank control: One blank control without test substance.
(5) Parallel: No replicates
(6) Number of test fish: 60 in total, 10 fish for each test group.
(7) The volume of test solution: 2 L / test vessel

Test environment
(1) The temperature of the test water:The temperature of each test vessel measured at regular intervals ranged from 22.2 ºC to 24.1 ºC, and the temperature detected continuously ranged from 22.2 ºC to 24.1 ºC during the test.
(2) pH: 7.53 – 8.38.
(3) Dissolved oxygen: 63% - 100%, not less than 60% of air saturation value.
(4) Light: Artificial light of white fluorescent lamp, 14 h light / 10 h darkness.

Feeding
The daily food ration was about 4% of the body weight of test fish, divided the food ration into two equal portions and given to the fish into two feeds per day, feeding interval was not less than 5 h. at 14 d after start of test, the body weight of test fish was reweighed, and the food ration was adjusted. In order to facilitate the weighing of test fish, at 0 d and 14 d was fed only once, the ration was about 4% of the body weight of test fish.

Observations and measurements
(1) Observation of the test fish: The fish were examined daily during the test period and any external abnormalities and mortalities were noted, the dead fish were removed as soon as possible.
(2) Hardness: Total hardness of blank control and 100 mg/L WAF test group were measured at the start of the test.
(3) Temperature, pH and dissolved oxygen: The temperature, pH and dissolved oxygen of all test groups were measured at 0 d, 7 d, 14 d, 21 d, 27 d (new solution) and 1 d, 8 d, 15 d, 22 d, 28 d (old solution). The temperature was continuously detected during the test period.
(4) Fish selection and weighing: At the beginning, 14d and 28d of the test, the test fish were weighed one by one.
(a) At 0 d after the start of the test (withheld from food for 24 h), fish were randomly chosen and put into 100 mg/L tricaine methane sulphonate (MS222) solution. Narcotized fish were dried with absorbent paper and then weighed one by one to the accuracy of 1 mg. Fish with a body weight ranged from 50 mg to 100 mg were put into a vessel that contained test water. All vessels were marked with test concentrations. After the test fish revived, they were transferred to the test solution.
(b) At 14 d (withheld from food for 24 h), all test fish were weighed again and the weight of the food was re-calculated.
(c) At 28 d (withheld from food for 24 h), the end of the test, all the test fish were anesthetized and weighed.

Analysis concentration of test substance
(1) Sampling: Test solutions of each test substance groups were collected at 0 d, 7 d, 14 d, 21 d (new solution) and 1 d, 8 d, 15 d, 22 d (old solution); test solution of blank control was collected at 0 d and 1 d (old solution), all the samples were used for analysis of test substance.
(2) Pre-treatment of sample: The test solutions were collected and filtered through 0.45 μm aqueous phase polyether sulfone (PES) filter membranes (the first 5 mL - 10 mL filtrate was discarded), 50 mL filtrate was transferred into separating funnel and extract three times with 20 mL, 15 mL and 10 mL of n-hexane, respectively. The three extracts were collected into 50 mL volume flask and diluted to 50 mL with n-hexane before analyzed.
(3) Storing condition: The samples were analyzed immediately, otherwise the samples were stored at 2 °C – 8 °C and protected from light after pretreatment, and the measurements were carried out within 96 h.
Reference substance (positive control):
not required
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Observation of test solutions
The WAF solution was prepared with the direct addition method. During the preparation periods, surface films in the top layers were observed in all test solutions, so the solution in the middle layer was siphoned into another test vessel as exposure solution, certainly, there no obvious droplets or surface films were observed in the exposure solution. Any remaining non-dissolved test chemical component in the WAF has the possibility to cause physical effects on the test organism unrelated to inherent chemical toxicity.

Measurement of pH, dissolved oxygen, temperature and total hardness of test solution
The pH, dissolved oxygen and temperature of all test groups were measured at 0 d, 7 d, 14 d, 21 d, 27 d (new solution) and 1 d, 8 d, 15 d, 22 d, 28 d (old solution), at the same time, the temperature was also detected continuously of water bath by a therometer during the test. The results showed that, pH values ranged from 7.53 to 8.38; the dissolved oxygen concentration ranged from 63% to 100% of the air saturation value (ASV), which met the requirement that the dissolved oxygen must have been at least 60% of ASV. The temperature of each test vessel measured at regular intervals ranged from 22.2 °C to 24.1 °C, which was within ± 1°C for all test vessels at each time of measurement. And the temperature detected continuously of water bath ranged from 22.2 °C to 24.1 °C during the test, which had satisfied with the requirement in the range of 2 °C.
At the start of the test, both of total hardness of blank control and 100 mg/L WAF test group were 115 mg/L (expressed as CaCO3).

The abnormal symptom of the test fish
At the end of the test, no abnormal symptoms and mortalities were observed in blank control, which met the requirement that the mortality should not exceed 10%. There also no abnormal symptoms and mortalities were found in the normal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups during the test period.

The weight of fish
At the end of the test, the mean weight of the blank control had increased by 116%, which met the criterion that the mean weight of fish in the control must have increased at least 50% of the initial weight over 28 days. The mean increased body weights of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were 128%, 118%, 102%, 119% and 110%, respectively.

Tank-average specific growth rate (r2) and “pseudo” specific growth rate (r3)
At the end of test, the average (r2) and standard deviation of r3 of blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were 2.72 ± 0.57 (d-1), 2.93 ± 0.46 (d-1), 2.80 ± 0.30 (d-1), 2.48 ± 0.63 (d-1), 2..78 ± 0.58 (d-1) and 2.65 ± 0.39 (d-1), respectively.

LOEL/ NOEL
The 28 d - NOEL of the juvenile growth test to Rare Minnow was the nominal loading rate of 100 mg/L (WAF), and the 28 d -LOEL of the juvenile growth test to Rare Minnow was greater than the nominal loading rate of 100 mg/L (WAF).

Analytical concentration of test substance in test solutions
The limit of detection (LOD) was 0.645 mg/L and limit of quantification (LOQ) was 2.15 mg/L.
The measured concentrations of test substance in blank control at 0 d and 1 d (old solution) were both lower than LOD of 0.645 mg/L.
The measured concentrations of test substance in nominal loading rate of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups at 0 d, 7 d, 14 d, 21 d (new solution) and 1 d, 8 d, 15 d, 22 d (old solution) were all lower than LOD of 0.645 mg/L. The measured concentrations of all test solutions lower than the LOD, this did not mean that there was no test substance present in the test solutions, just that the concentration was below the LOD of the analytical method, indicating that test substance was poorly soluble in test water.

Validity of the test
(1) At the end of the test, the mortality of the blank control was 0%, not exceeded 10%.
(2) The mean body weight of fish in the blank control had increased by 116%, and thus increased by at least 50% of the mean initial weight over 28 days.
(3) The dissolved oxygen of test water though the test ranged from 63%-100% of the air saturation value (ASV) and met the requirement of at least 60% of ASV.
(4) The temperature of water bath detected continuously ranged from 22.2 ºC to 24.1 ºC during the test, which had satisfied with the requirement in the range of 2 °C. The temperature of each test vessel detected at regular intervals within ± 1°C for all test vessels at each time of measurement.
Results with reference substance (positive control):
Not required
Reported statistics and error estimates:
The “pseudo” specific growth rate r3 conform to the normal distribution and the homogeneous of variance, so the Dunnett’s test (< control) was used to estimate the NOEL and LOEL. Results of significant test (p>0.05) showed that, there were no significant different on r3 between the blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups.

The pH of the test solution during the test

Nominal loading rate

0 d

1 d

7 d

8 d

14 d

15 d

21 d

22 d

27 d

28 d

N

O

N

O

N

O

N

O

N

O

Blank control

8.30

7.88

7.95

7.70

8.25

7.55

8.32

7.79

8.17

7.54

1.0 mg/L WAF

8.37

7.88

8.30

7.75

8.33

7.55

8.37

7.80

8.24

7.53

3.2 mg/L WAF

8.35

7.89

8.33

7.76

8.34

7.55

8.38

7.79

8.23

7.54

10 mg/L WAF

8.37

7.87

8.35

7.78

8.35

7.55

8.36

7.78

8.24

7.56

32 mg/L WAF

8.34

7.88

8.33

7.77

8.34

7.53

8.36

7.78

8.25

7.54

100 mg/L WAF

8.32

7.88

8.33

7.75

8.35

7.55

8.36

7.79

8.25

7.56

Note: “N” means new solution, “O” means old solution.

 

The dissolved oxygen of the test solution during the test (%)

Nominal loading rate

0 d

1 d

7 d

8 d

14 d

15 d

21 d

22 d

27 d

28 d

N

O

N

O

N

O

N

O

N

O

Blank control

98

78

95

65

99

65

95

69

100

67

1.0 mg/L WAF

98

77

100

66

100

63

95

68

100

65

3.2 mg/L WAF

99

79

100

68

100

65

95

69

100

66

10 mg/L WAF

100

78

99

67

100

65

95

66

100

65

32 mg/L WAF

100

78

99

68

100

66

96

66

100

67

100 mg/L WAF

99

78

100

67

100

65

96

65

100

65

Note: “N” means new solution, “O” means old solution.

 

The water temperature of the test solution during the test (°C)

Nominal loading rate

0 d

1 d

7 d

8 d

14 d

15 d

21 d

22 d

27 d

28 d

N

O

N

O

N

O

N

O

N

O

Blank control

24.0

23.1

23.9

24.1

23.8

23.7

22.9

22.5

22.5

22.3

1.0 mg/L WAF

24.0

23.1

23.9

24.0

23.8

23.6

22.8

22.5

22.5

22.4

3.2 mg/L WAF

24.0

23.2

23.9

23.9

23.8

23.6

22.8

22.5

22.5

22.4

10 mg/L WAF

24.0

23.1

23.9

24.0

23.8

23.6

22.8

22.5

22.3

22.3

32 mg/L WAF

24.0

23.1

24.0

24.0

23.8

23.7

22.8

22.5

22.3

22.3

100 mg/L WAF

24.0

23.1

24.0

24.0

23.8

23.6

22.8

22.5

22.3

22.2

Note: “N” means new solution, “O” means old solution.

 

The measure concentrations of test substance

Nominal loading rate

Measured concentration (mg/L)

0 d N

1 d O

7 d N

8 d O

14 d N

15 d O

21 d N

22 d O

Blank control

<LOD

<LOD

-

-

-

-

-

-

1.0 mg/L WAF

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

3.2 mg/L WAF

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

10 mg/L WAF

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

32 mg/L WAF

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

100 mg/L WAF

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

Note: Limit of detection (LOD) and limit of quantification (LOQ) of test substance were 0.645 mg/ and 2.15 mg/L, respectively, “<LOD” means lower than the LOD “-“ means not detected.

 

The abnormal symptoms during the exposed period

Nominal loading rate

1 d

2 d

3 d

4 d

5 d

6 d

7 d

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Blank control

0

N

0

N

0

N

0

N

0

N

0

N

0

N

1.0 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

3.2 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

10 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

32 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

100 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

Note:“N” means normal

The abnormal symptoms during the exposed period (continued)

Nominal loading rate

8 d

9 d

10 d

11 d

12 d

13 d

14 d

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Blank control

0

N

0

N

0

N

0

N

0

N

0

N

0

N

1.0 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

3.2 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

10 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

32 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

100 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

Note:“N” means normal

The abnormal symptoms during the exposed period (continued)

Nominal loading rate

15 d

16 d

17 d

18 d

19 d

20 d

21 d

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Blank control

0

N

0

N

0

N

0

N

0

N

0

N

0

N

1.0 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

3.2 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

10 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

32 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

100 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

Note:“N” means normal

The abnormal symptoms during the exposed period (continued)

Nominal loading rate

22 d

23 d

24 d

25 d

26 d

27 d

28 d

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Number of death

Abnormal symptom

Blank control

0

N

0

N

0

N

0

N

0

N

0

N

0

N

1.0 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

3.2 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

10 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

32 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

100 mg/L WAF

0

N

0

N

0

N

0

N

0

N

0

N

0

N

Note:“N” means normal

 

The weight of fish during the test period

Time

Nominal loading rate

Weight of individual fish (mg)

M±SD (mg)

ΔW (%)

1

2

3

4

5

6

7

8

9

10

0 d

Blank control

58

66

63

61

63

52

64

65

56

54

60±5

-

1.0 mg/L WAF

64

56

62

64

55

61

62

51

66

54

60±6

-

3.2 mg/L WAF

56

68

60

54

51

70

62

52

61

70

60±8

-

10 mg/L WAF

64

61

69

51

62

54

52

66

61

58

60±6

-

32 mg/L WAF

63

54

69

70

56

66

58

52

51

56

60±7

-

100 mg/L WAF

67

69

55

58

56

69

58

57

58

55

60±6

-

14 d

Blank control

69

74

73

64

69

64

55

78

61

66

67±7

-

1.0 mg/L WAF

73

73

64

74

68

80

62

76

58

64

69±8

-

3.2 mg/L WAF

77

80

78

65

70

72

65

64

75

64

71±7

-

10 mg/L WAF

80

64

72

85

70

73

57

69

69

69

71±8

-

32 mg/L WAF

69

81

64

75

61

82

59

78

58

69

70±10

-

100 mg/L WAF

80

63

84

61

82

69

64

62

65

75

70±9

-

28 d

Blank control

179

120

135

126

144

138

112

116

131

100

130±22

116

1.0 mg/L WAF

132

129

143

119

145

117

140

164

155

111

136±18

128

3.2 mg/L WAF

125

144

121

140

127

115

125

133

150

137

132±12

118

10 mg/L WAF

133

95

136

160

120

131

93

99

120

120

121±21

102

32 mg/L WAF

131

115

109

113

171

160

123

148

125

109

130±23

119

100 mg/L WAF

105

128

120

139

105

144

133

133

133

125

126±14

110

Note: “M” means the average fish weight, “SD” mean standard deviation. “ΔW” means the percentage of increased weight relative to initial fish weight.

 

The r2and r3value at the end of the test

Nominal loading rate

r3(d-1)

r2(d-1)

1

2

3

4

5

6

7

8

9

10

M±SD

Blank control

3.90

2.47

2.90

2.65

3.13

2.97

2.23

2.35

2.79

1.82

2.72±0.57

1.0 mg/L WAF

2.86

2.78

3.14

2.49

3.19

2.43

3.07

3.63

3.43

2.24

2.93±0.46

3.2 mg/L WAF

2.62

3.13

2.50

3.03

2.68

2.32

2.62

2.84

3.27

2.95

2.80±0.30

10 mg/L WAF

2.87

1.67

2.95

3.53

2.50

2.82

1.59

1.82

2.50

2.50

2.48±0.63

32 mg/L WAF

2.84

2.38

2.18

2.31

3.79

3.55

2.62

3.28

2.67

2.18

2.78±0.58

100 mg/L WAF

2.00

2.71

2.48

3.00

2.00

3.13

2.85

2.85

2.85

2.62

2.65±0.39

NOEL

= 100 mg/L (WAF)

LOEL

> 100 mg/L (WAF)

Note:

The “pseudo” specific growth rate (r3) expresses the individual growth rate compared to the mean initial weight of the tank population.

“M” mean the average growth rate, equal to the test vessel growth rate r2, “SD” mean the standard deviation of r3.

Validity criteria fulfilled:
yes
Conclusions:
The definitive test was conducted under semi-static condition (renew solutions every 24 h), at the end of test, no test fish were died in blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups.
At the end of test, the average and standard deviation of "pseudo" specific growth rate (r3) of test fish in blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were 2.72 ± 0.57 (d-1), 2.93 ± 0.46 (d-1), 2.80 ± 0.30 (d-1), 2.48 ± 0.63 (d-1), 2.78 ± 0.58 (d-1) and 2.65 ± 0.39 (d-1), respectively. Results of significant test (Dunnett’s test, < control) showed that there were no significant different (p>0.05) on r3 between the blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups. Hence, the 28 d - NOEL of juvenile growth test to Rare Minnow was the nominal loading rate of 100 mg/L (WAF), and the 28 d -LOEL of juvenile growth test to Rare Minnow was greater than the nominal loading rate of 100 mg/L (WAF).
Executive summary:

Purpose of the test

This test guideline was designed to assess the effects of prolonged exposure to chemicals on the growth of juvenile fish.

 

Guidelines

[1] State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, No. 215 Fish, Juvenile Growth Test. Second Edition. Beijing: China Environmental Science Press, 2013: 135-144.

[2] China National Technical Committee for Standardization of Dangerous Chemical Management GBT 21806-2008. Chemical Fish, Juvenile Growth Test [S]. Beijing: China Standardization Press, 2008.

[3] OECD. OECD Guidelines for Testing of Chemicals, No. 215 Fish, Juvenile Growth Test[S]. OECD: Adopted 21 January, 2000.

[4] OECD. OECD series on testing and assessment number 23 (Second Edition), Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals. Paris: OECD, Adopted on February 8, 2019.

[5] ECETOC. Aquatic Toxicity Testing of Sparingly Soluble, Volatile and Unstable Substances [S]. ECETOC Monograph No. 26 1996.

[6] ASTM international. Standard Practice for Aquatic Toxicity Testing of Lubricants: Sample Preparation and Results Interpretation. D6081 − 98 (Reapproved 2014).

[7] State Environmental Protection Administration of China. HJ/T 153-2004. The Guidelines for the test of chemical [S]. Beijing: China Environmental Science Press. 2004.

 

Principle

Juvenile fish in exponential growth phase are placed, after being weighed, in test chambers and are exposed to a range of sublethal loading rates of the test substance dissolved in water preferably under semi static conditions. The test duration is 28 days. Fish are fed daily. The food ration is based on initial fish weights and recalculated after 14 days. At the end of the test, the fish are weighed again. Effects on growth rates are analyzed, the data may be compared with control values in order to determine the LOEL and NOEL.

 

Test conditions

(1) Test substance: SynNova Base Oil

(2) Test organism: Rare Minnow (Gobiocypris rarus)

(3) Exposure duration: 28 d

(4) Test concentration: According to the results of preliminary tests (see Annex C), the definitive test was conducted with one blank control and five nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L Water Accommodate Fraction (WAF) test groups.

(5) Replicate: No replicates

(6) Number of fish: 60 in total (10 fish for each group)

(7) Test water: Dechlorination tap water

(8) Type of test: Semi-static test with renewals at 24 h intervals.

(9) Preparation of the test solution: 5.0 mg, 16 mg, 50 mg, 160 mg and 500 mg test substance were respectively weighed into flasks and mixed with small amount of dechlorinated tap water, then the mixtures were transferred intovessels and diluted with dechlorinated tap water to 5000 mL respectively, the nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L solutions were obtained. The solutions were stirred for about 24 h (the depth of vortex was maintained at one third of the test solution height) and stood for about 1 h, then 2000 mL of middle layer solutions were collected into anothervessels (the first 100 – 200 mL solution was discarded), the nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF solutions were obtained and used as test solutions.This procedure is in accordance with the OECD guidance document No. 23 (2019) and ASTM D6081 (2014).

(10) Volume of test solution: 2 L / test vessel

(11) Temperature of test solutions: The temperature of all test vessels detected regularly intervals ranged from 22.2 ºC to 24.1 ºC, and the temperature detected continuously ranged from 22.2 ºC to 24.1 ºC during the test.

(12) Photoperiod: 14 h light / 10 h darkness

(13) Feeding: The daily food ration was about 4% of the body weight of test fish. The daily ration was divided into two equal portions and given to the fish in two feeds per day, separated by at least 5 hours. At 14 d after start of test, the body weight of test fish was reweighed and the food ration was adjusted. In order to facilitate the weighing of test fish, at 0 d and 14 d, the test fish were feed only once, the ration was about 4% of the body weight of test fish.

(14) Analysis of the actual concentration of test substance in test solutions: The actual concentrations of test substance in 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were measured at 0 d, 7 d, 14 d, 21 d (new solution) and 1 d, 8 d, 15 d, 22 d (old solution) by Gas Chromatograph (GC); the blank control was measured at 0 d (new solution) and 1 d (old solution).

 

Results

(1) Basis of preparation test solution

The test substance SynNova Base Oil is Liquid - water white colorless oil. The test substance is a complex mixture / UVCB substance (Substances of Unknown or Variable Composition, Complex Reaction Products or Biological Materials) and is poorly soluble in test water. The aqueous media were prepared following ASTM D6081-98/Reapproved 2014), which is specifically developed for aquatic toxicity testing of lubricants, by mixing the test substance with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the WAF was separated by siphon. The WAF was checked for any undissolved or emulsified material by microscopic inspection, which was negative. Exposure was expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate). This procedure was followed for each renewal of the test solutions.

(2) Measured concentrations of test substance in test solution

The results of the analytical methods validation of test substance are presented in the final report of “Validation of an Analytical Method of SynNova Base Oil” (The report No. is A19058-T19054-1R). The limit of detection (LOD) was 0.645 mg/L and limit of quantification (LOQ) was 2.15 mg/L.

The measured concentrations of test substance in blank control at 0 d and 1 d (old solution) were both lower than LOD of 0.645 mg/L.

The measured concentrations of test substance in nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups at 0 d, 7 d, 14 d, 21 d (new solution) and 1 d, 8 d, 15 d, 22 d (old solution) were all lower than LOD of 0.645 mg/L. The measured concentrations of all test solutions lower than the LOD, this did not mean that there was no test substance present in the test solutions, just that the concentration was below the LOD of the analytical method, indicating that test substance was poorly soluble in test water.

(3) Toxicity effects on test organism

The definitive test was conducted under semi-static condition (renew solutions every 24 h), at the end of test, no test fish were died in blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups.

At the end of test, the average and standard deviation of "pseudo" specific growth rate (r3) of test fish in blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were 2.72 ± 0.57 (d-1), 2.93 ± 0.46 (d-1), 2.80 ± 0.30 (d-1), 2.48 ± 0.63 (d-1), 2.78 ± 0.58 (d-1) and 2.65 ± 0.39 (d-1), respectively. Results of significant test (Dunnett’s test, < control) showed that, there were no significant different (p>0.05) on r3 between the blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups. Hence, the 28 d - No Observed Effect Loading rate (NOEL) of juvenile growth test to Rare Minnow was the nominal loading rate of 100 mg/L (WAF), and the 28 d - Lowest Observed Effect Loading rate (LOEL) of juvenile growth test to Rare Minnow was greater than the nominal loading rate of 100 mg/L (WAF).

Description of key information

OECD 210 -Guangdong Detection Center of Microbiology-2019ESG0172

Hatching rate: NOEL =100mg/L; LOEL >100mg/L

Surviving rate after post-hatched: NOEL =100mg/L; LOEL >100mg/L

Cumulative abnormal rate: NOEL =100mg/L; LOEL >100mg/L

Cumulative mortality: NOEL =100mg/L; LOEL >100mg/L

Total length of surviving fish: NOEL =100mg/L; LOEL >100mg/L

Dry weight of surviving fish: NOEL =100mg/L; LOEL >100mg/L

 

OECD 212 -Guangdong Detection Center of Microbiology-2019ESG0173

Hatching rate: NOEL =100 mg/L; LOEL >100 mg/L

Surviving rate of larvae after post-hatched: NOEL =100 mg/L; LOEL >100 mg/L

Cumulative abnormal rate of embryos/larvae: NOEL =100 mg/L; LOEL >100 mg/L

Cumulative mortality of embryos/larvae: NOEL =100 mg/L; LOEL >100 mg/L

Total length of surviving larvae: NOEL =100 mg/L; LOEL >100 mg/L

Dry weight of surviving larvae: NOEL =100 mg/L; LOEL >100 mg/L

OECD 215 -Guangdong Detection Center of Microbiology-2019ESG0174

28 d - No Observed Effect Loading rate (NOEL) of juvenile growth test to Rare Minnow was the nominal loading rate of 100 mg/L (WAF), and the 28 d - Lowest Observed Effect Loading rate (LOEL) of juvenile growth test to Rare Minnow was greater than the nominal loading rate of 100 mg/L (WAF).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

Additional information

OECD 210 -Guangdong Detection Center of Microbiology-2019ESG0172

Tests with the early-life stages of fish are intended to define the lethal and sub-lethal effects of chemicals on the stages and species tested. They yield information of value for the estimation of the chronic lethal and sub-lethal effects of the chemical on other species.

Results

(1) Basis of preparation test solution

The test substance SynNova Base Oil is liquid - water white colorless oil. The test substance is a complex mixture / UVCB substance (Substances of Unknown or Variable Composition, Complex Reaction Products or Biological Materials) and is poorly soluble in test water. The aqueous media were prepared following ASTM D6081-98/Reapproved 2014), which is specifically developed for aquatic toxicity testing of lubricants, by mixing the test substance with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the WAF was separated by siphon. The WAF was checked for any undissolved or emulsified material by microscopic inspection, which was negative. Exposure was expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate). This procedure was followed for each renewal of the test solutions.

(2) The measured concentrations of test substance in test solutions

The results of the analytical methods are shown in the final report of “Validation of an Analytical Method of SynNova Base Oil” (The report No. is A19058-T19054-1R). Limit of detection (LOD) and limit of quantification (LOQ) of analytical method were 0.645 mg/L and 2.15 mg/L, respectively.

The measured concentrations of test substance in the blank control at 0 d (new solution) and 1 d (old solution) were both lower than LOD of 0.645 mg/L.

The measured concentrations of test substance in nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d, 33 d (new solution) and 1 d, 8 d, 11 d, 15 d, 22 d, 28 d, 34 d (old solution) were all lower than LOD of 0.645 mg/L. The measured concentrations of all test solutions lower than the LOD, this did not mean that there was no test substance present in the test solutions, just that the concentration was below the LOD of the analytical method, indicating that test substance was poorly soluble in test water.

(3) Toxicity effects on test organism

The test substance is a kind of UVCB item, and insoluble in test water, so water accommodate fraction was used to prepare test solution and the test results were expressed as the nominal loading rate. Under this test condition, at the end of the test (34 d), the NOEL and LOEL of Fish, Early-life Stage Toxicity Test to Rare Minnow (Gobiocypris rarus) are shown as follow:

Hatching rate: NOEL =100mg/L; LOEL >100mg/L

Surviving rate after post-hatched: NOEL =100mg/L; LOEL >100mg/L

Cumulative abnormal rate: NOEL =100mg/L; LOEL >100mg/L

Cumulative mortality: NOEL =100mg/L; LOEL >100mg/L

Total length of surviving fish: NOEL =100mg/L; LOEL >100mg/L

Dry weight of surviving fish: NOEL =100mg/L; LOEL >100mg/L

OECD 212 -Guangdong Detection Center of Microbiology-2019ESG0173

The embryo and sac-fry stages of fish are exposed to a range of concentrations of the test substance dissolved in water under semi-static conditions. The test is begun by placing fertilized eggs in the test chambers and is terminated just before the yolk-sac of any larvae in any of the test chambers has been completely absorbed or before mortalities by starvation start in controls. Lethal and sub-lethal effects are assessed and compared with control values to determine the lowest observed effect loading rate (LOEL) and hence the no observed effect loading rate (NOEL).

 

Results

(1) Basis of preparation test solution

The test substance SynNova Base Oil is liquid - water white colorless oil. The test substance is a complex mixture / UVCB substance (Substances of Unknown or Variable Composition, Complex Reaction Products or Biological Materials) and is poorly soluble in test water. The aqueous media were prepared following ASTM D6081-98/Reapproved 2014), which is specifically developed for aquatic toxicity testing of lubricants, by mixing the test substance with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the WAF was separated by siphon. The WAF was checked for any undissolved or emulsified material by microscopic inspection, which was negative. Exposure was expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate). This procedure was followed for each renewal of the test solutions.

(2) The measured concentrations of test substance in test solutions

The results of the analytical methods are shown in the final report of “Validation of an Analytical Method of SynNova Base Oil” (The report No. is A19058-T19054-1R). Limit of detection (LOD) and limit of quantification (LOQ) of analytical method were 0.645 mg/L and 2.15 mg/L, respectively.

The measured concentrations of test substance in the blank control at 0 d (new solution) and 1 d (old solution) were both lower than LOD of 0.645 mg/L.

The measured concentrations of test substance in 100 mg/L WAF test group at 0 d, 3 d, 7 d (new solution) and 1 d, 4 d, 8 d (old solution) were all lower than LOD of 0.645 mg/L. The measured concentrations of all test solutions lower than the LOD, this did not mean that there was no test substance present in the test solutions, just that the concentration was below the LOD of the analytical method, indicating that test substance was poorly soluble in test water.

(3) Toxicity effects on test organism

The test substance is a kind of UVCB item, and insoluble in test water, so water accommodate fraction was used to prepare test solution and the test results were expressed as the nominal loading rate. Under this test condition, at the end of the test (9 d), the No Observed Effect loading rate (NOEL) and Lowest Observed Effect loading rate (LOEL) to embryos of Rare Minnow (Gobiocypris rarus) are shown as follow:

Hatching rate: NOEL =100 mg/L; LOEL >100 mg/L

Surviving rate of larvae after post-hatched: NOEL =100 mg/L; LOEL >100 mg/L

Cumulative abnormal rate of embryos/larvae: NOEL =100 mg/L; LOEL >100 mg/L

Cumulative mortality of embryos/larvae: NOEL =100 mg/L; LOEL >100 mg/L

Total length of surviving larvae: NOEL =100 mg/L; LOEL >100 mg/L

Dry weight of surviving larvae: NOEL =100 mg/L; LOEL >100 mg/L

OECD 215 -Gunagdong Detection Center of Microbiology-2019ESG0174

Juvenile fish in exponential growth phase are placed, after being weighed, in test chambers and are exposed to a range of sublethal loading rates of the test substance dissolved in water preferably under semi static conditions. The test duration is 28 days. Fish are fed daily. The food ration is based on initial fish weights and recalculated after 14 days. At the end of the test, the fish are weighed again. Effects on growth rates are analyzed, the data may be compared with control values in order to determine the LOEL and NOEL.

 

(1) Basis of preparation test solution

The test substance SynNova Base Oil is Liquid - water white colorless oil. The test substance is a complex mixture / UVCB substance (Substances of Unknown or Variable Composition, Complex Reaction Products or Biological Materials) and is poorly soluble in test water. The aqueous media were prepared following ASTM D6081-98/Reapproved 2014), which is specifically developed for aquatic toxicity testing of lubricants, by mixing the test substance with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the WAF was separated by siphon. The WAF was checked for any undissolved or emulsified material by microscopic inspection, which was negative. Exposure was expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate). This procedure was followed for each renewal of the test solutions.

(2) Measured concentrations of test substance in test solution

The results of the analytical methods validation of test substance are presented in the final report of “Validation of an Analytical Method of SynNova Base Oil” (The report No. is A19058-T19054-1R). The limit of detection (LOD) was 0.645 mg/L and limit of quantification (LOQ) was 2.15 mg/L.

The measured concentrations of test substance in blank control at 0 d and 1 d (old solution) were both lower than LOD of 0.645 mg/L.

The measured concentrations of test substance in nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups at 0 d, 7 d, 14 d, 21 d (new solution) and 1 d, 8 d, 15 d, 22 d (old solution) were all lower than LOD of 0.645 mg/L. The measured concentrations of all test solutions lower than the LOD, this did not mean that there was no test substance present in the test solutions, just that the concentration was below the LOD of the analytical method, indicating that test substance was poorly soluble in test water.

(3) Toxicity effects on test organism

The definitive test was conducted under semi-static condition (renew solutions every 24 h), at the end of test, no test fish were died in blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups.

At the end of test, the average and standard deviation of "pseudo" specific growth rate (r3) of test fish in blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were 2.72 ± 0.57 (d-1), 2.93 ± 0.46 (d-1), 2.80 ± 0.30 (d-1), 2.48 ± 0.63 (d-1), 2.78 ± 0.58 (d-1) and 2.65 ± 0.39 (d-1), respectively. Results of significant test (Dunnett’s test, < control) showed that, there were no significant different (p>0.05) on r3 between the blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups. Hence, the 28 d - No Observed Effect Loading rate (NOEL) of juvenile growth test to Rare Minnow was the nominal loading rate of 100 mg/L (WAF), and the 28 d - Lowest Observed Effect Loading rate (LOEL) of juvenile growth test to Rare Minnow was greater than the nominal loading rate of 100 mg/L (WAF).