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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 June 2016 - 22 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Solubility in water: predicted to be between 1 and 10 mg/L
Stability in water: not indicated
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from the highest concentration (i.e. undiluted WAF) and the control.
Frequency: at the beginning and at the end of four intervals of 48 hours (nominal days 0 and 2, 4 and 6, 12 and 14, 18 and 20).
Volume: 16 mL
Storage: Samples not analysed on the day of sampling were stored in a freezer until analysis.

Additionally, reserve samples of 16 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: a Water Accommodated Fraction (WAF) was prepared at 1.0 mg/L applying a 24-hours period of magnetic stirring after which the resulting mixtures were allowed to stabilize for 1 hour. Subsequently, the aqueous phase was siphoned off over glass wool and used as the highest concentration. The lower test concentrations were prepared by diluting the highest concentration in test medium. All final test solutions were clear and colorless. This approach was used because it was not feasible to prepare loading rates as low as required for the full test. Moreover, this method allowed for calculation of the lower exposure concentrations that could not be measured with the available analytical method. It should be noted that development of a more sensitive method was not feasible.
- Controls: test medium without test item or other additives.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: in-house laboratory culture with a known history. Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Age of parental stock: > 2 weeks old
- Feeding during test: once per day an amount of 0.5 ml of a Chlorella sp. suspension, corresponding to 0.2 mg C/Daphnia/day.

BREEDING:
- Stat of each batch: with newborn daphnids, i.e. less than 3 days old, by placing them individually in 50 ml M7-medium.
- Breeding conditions: same as test
- Feeding: daily, a suspension of fresh water algae
- Health: no signs of stress such as mortality was observed
- Acclimation period: no
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
179 - 196 mg CaCO3/L
Test temperature:
19 - 20 °C
pH:
7.3 - 8.6
Dissolved oxygen:
6.4 - 9.9 mg/L
Nominal and measured concentrations:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test:
Nominal: 1.0, 3.2, 10, 32 and 100% of WAF prepared at 1.0 mg/L
Measured (1.0 mg/L): fresh solutions at day 0: 0.180 mg/L, day 4: 0.218 mg/L, day 12: 0.365 mg/L, day 28: 0.0676 mg/L; old solutions at day 2: 0.156 mg/L, day 6: 0.194 mg/L, day 14: 0.178 mg/L, day 20: 0.0675
Geometric mean was calculated as: 0.168 mg/L (d 0-2), 0.206 mg/L (d 4-6), 0.255 mg/L (d 12-14) and 0.0675 mg/L (d 18-20).
The average exposure concentration was calculated as: 0.17 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 mL, all-glass with headspace reduced to a minimum and closed air tight
- Aeration: no
- Renewal rate of test solution: every 48 hours
- No. of organisms per vessel: 1 neonate daphnid
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 20

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water purified by Reverse Osmosis (RO-water)
- Culture medium different from test medium: no, M7
- Intervals of water quality measurement: at the start of the test and just before and after each renewal (every 48 hours)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h photoperiod daily
- Light intensity: intensity at the start: 554-576 lux, intensity at the end: 499-719 lux

EFFECT PARAMETERS MEASURED: EC50, NOEC and LOEC for reproduction, mortality and growth

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Test concentrations for range finding study: 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes
- In addition, a stability test was performed to asses the stability of the test item during a 48 hour period. A WAF prepared at 1.0 mg/L was incubated with daphnia and algae for 48 hours under test conditions. Samples were taken at the start, after 24 and 48 hours of exposure. Three replicate vessels were used. Results showed that concentrations were sufficiently stable over the 48 hour period and could therefore be renewed using this time interval.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.053 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
reproduction
Remarks on result:
other: Effect concentration was calculated based on the average exposure concentration times the used dilution factor (i.e. 3.2)
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
> 0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.053 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth
Remarks on result:
other: Effect concentration was calculated based on the average exposure concentration times the used dilution factor (i.e. 3.2).
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth
Details on results:
- Reproduction and growth were statistically significantly reduced at the highest test concentration
- Three out of the twenty parental daphnids died during the test period in the control. Hence, parental mortality did not exceed 20% in the control. The mortality observed in this test ranged from 10 to 30% and was not related to the applied dose or statistically significant from the control treatment. Therefore, according to the guideline, the effect parameters were calculated based on the surviving animals.
- Actual concentrations measured in the freshly prepared WAF ranged from 0.068 to 0.37 mg/L. Measured concentrations were at the level of 49 to 100% of initial at the end of the refreshment period. The calculated average exposure concentration was 0.17 mg/L. The lower concentrations were not sampled as it was known that they could not be quantified with the available analytical method. Therefore, the lower exposure concentrations were calculated based on the used dilution factor (i.e. 3.2) (for details on measured concentrations see Table 1 in 'Any other information on results').
- All test conditions (dissolved oxygen concentrations, pH, temperature, hardness and total dissolved organic carbon) were maintained within the limits prescribed by the study plan.
-Three reproduction tests were performed with a daily renewal of test solutions. In all these tests the validity criterion of at least 80% survival in the control treatment was not met and therefore, all of them were considered not valid. Decided was to conduct a final test with refreshment every 48 hour. Before this decision was taken an additional stability test was performed. The stability test showed measured concentrations of 77-96% of initial after 24 hours and 69-89% of initial after 48 hours. This showed that the concentrations were sufficiently stable over a period of 48 hours.
Reported statistics and error estimates:
Following statistical procedures were used to determine the NOEC for reproduction and growth:
- Data distribution: Shapiro-Wilk´s Test
- Homogeneity of variance: Levene´s Test (with Residuals)
Differences between treatments and the control: Multiple Sequentially-rejective U-test after Bonferroni-Holm (reproduction) and Step-down Jonckheere-Terpstra Test Procedure (growth), both one-side smaller, alpha = 0.050. Mortality of parental daphnids was analysed with Fisher`s Exact Binomial Test with Bonferroni Correction (alpha = 0.050, one-sided greater).

All analyses were performed with ToxRat Professional 3.2.1 (ToxRat Solutions® GmbH, Germany).

Table 1 Measured and average exposure concentrations in WAF prepared at 1.0 mg/L

Nominal day

Solution

Concentration (mg/L)

Measured

Geometric mean

Average exposure

0

(fresh)

0.180

0.168

0.17

2

(old)

0.156

4

(fresh)

0.218

0.206

6

(old)

0.194

12

(fresh)

0.365

0.255

14

(old)

0.178

18

(fresh)

0.0676

0.0675

20

(old)

0.0675

It should be noted that, although the preparation method was similar during each interval, it yielded different initial results. This was most probably caused by the fact that the test item was poorly soluble in test medium and every small difference in the preparation method could affect the result. However, in the first three intervals the measured concentrations decreased to a similar level (176 ± 0.019 µg/L, mean ± standard deviation). This suggests that the initial concentrations were above solubility limit. In contrary, during the last interval, concentrations remained stable, suggesting that they were below the solubility limit.

Table 2 Calculated exposure concentrations in the reproduction test

% of WAF prepared at 1.0 mg/L

Exposure concentration (mg/L)

100

0.17*

32

0.053#

10

0.017#

3.2

0.0053#

1.0

0.0017#

*- average exposure concentration determined based on measured values
# - calculated based on dilution factor of 3.2

Table 3 Mortality (immobility) of parental daphnids at 21 of exposure

Concentration test item (mg/L)

Introduced

Mobile

Immobile

% Immobility

Control

20

17

3

15

0.0017

10

7

3

30

0.0053

10

8

2

20

0.017

10

8

2

20

0.053

10

9

1

10

0.17

10

9

1

10

Table 4 Group mean cumulative number of juveniles per surviving parent and reduction of reproduction at day 21 of exposure

Concentration test item (mg/L)

Mean

Std. Dev.

n

% Reduction

Control

126.9

12.21

17

 

0.0017

137.4

6.35

7

-8.3

0.0053

121.1

44.30

8

4.6

0.017

129.4

44.49

8

-1.9

0.053

136.8

6.63

9

-7.7

0.17

92.3*

42.54

9

27.3

* - effect statistically significant

Table 5 Group mean body lengths (mm) and reduction of growth of parental daphnids at day 21 of exposure 

Test item concentration

(mg/L)

Mean

(mm)

Std. Dev.

n

%Reduction

Control

4.45

0.157

17

 

0.0017

4.42

0.062

7

0.7

0.0053

4.34

0.581

8

2.5

0.017

4.29

0.494

8

3.6

0.053

4.36

0.124

9

2.0

0.17

3.85*

0.441

9

13.5

Validity criteria fulfilled:
yes
Remarks:
1) The mortality of the parent animals (female Daphnia) in the control did not exceed 20% at the end of the test (i.e. 15%). 2) The average cumulative number of young per female in the controls after 21 days was ≥ 60 (i.e. 127 ± 9.6% CV).
Conclusions:
Under the conditions of the present study the test item did not affect reproduction and growth of Daphnia magna at 0.053 mg/L after 21 days of exposure (NOEC).
Exposure to mean exposure concentrations of 0.17 mg/L induced significant inhibition of the reproductive capacity of the parental daphnids.
Executive summary:

An acute Daphnia was not performed due to the toxicity seen and the low water solubility of the substance in the test media in a preliminary test. Therefore a long-term toxicity according to OECD TG 211 test was performed. In accordance with OECD Guidance 23 (2000), water accommodated fractions (WAFs) of the substance were used. In a screening test toxicity was seen. No mortality was observed in the lowest WAF of 1 mg/l, whereas all daphnids exposed to WAFs prepared at 10 and 100 mg/L were found dead after 24 hours of exposure. Therefore, in the main test a WAF was prepared at 1.0 mg/L and stirred for 24 hours. Subsequently, the aqueous phase was siphoned off over glass wool to separate undissolved residue and used as the highest concentration. The lower test concentrations were prepared by diluting the highest concentration in test medium. This is in line with the OECD Guidance 23 where it says that at low concentrations dilutions should be used instead of preparing separate solutions of the testing concentrations. During preliminary testing it was observed that the Daphnia were sensitive about being transferred to new solutions. Based on further stability testing the substance was stable in test medium for 48 hours (69 -89% of initial). Therefore, test solutions in the main test were renewed every 48 hours.

Following a preliminary range-finding test in which toxicity was seen, in the main test parental Daphnia magna were exposed to solutions of the test material at nominal concentrations of 1.0, 3.2, 10, 32 and 100% of WAF prepared at a loading rate of 1.0 mg/L (one replicate and one parental Daphnia per concentration) for 21 days, under semi-static conditions. During the study samples for analyses were taken from the undiluted WAF at the beginning and the end of four intervals of 48 hours. Actual concentration measured in the freshly prepared 100% WAF ranged from 0.068 to 0.37 mg/L. Measured concentrations were at the level of 49 to 100% of initial at the end of the 48 h refreshment period. The calculated average exposure concentration was 0.17 mg/L (100% WAF). The lower exposure concentrations were calculated based on the dilution factor of 3.2 and corresponded to 0.0017, 0.0053, 0.017 and 0.053 mg/L in solutions containing 1.0, 3.2, 10, 32% WAF, respectively.

In the control, 15% mortality of adult animals was observed. Therefore, in test solutions mortality between 10 and 30% was considered not related to the applied dose.

In the control, 127 offspring were produced per surviving daphnid. At 0.0017, 0.0053, 0.017 and 0.053 mg/l (1, 3.2, 10 and 32% of the WAF) there were no effects on reproduction, growth and mortality. At 0.17 mg/l effects were seen in reproduction and growth: Reproduction decreased by 27.3% and growth with 13.5%. Mortality at this concentration (as well as at the highest concentration of 0.17 mg/l) was not affected, and the EC50 values were not reached > 0.17 mg/l.

A significant reduction of reproduction (as calculated per surviving daphnid) was observed in the highest group (27% reduction). There was no delay in the onset of reproduction. The body length of daphnids exposed to the highest concentration was significantly reduced at the end of the test (13% reduction). Under the conditions of the present study the test item did not affect reproduction and growth of Daphnia magna at 0.053 mg/L after 21 days of exposure (NOEC). Exposure to mean exposure concentrations of 0.17 mg/L induced significant inhibition of the reproductive capacity of the parental daphnids. The study met acceptable criteria.

Description of key information

Long-term Daphnia, according to OECD TG 211: NOEC is 0.053 mg/l (based on reproduction and on growth).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.053 mg/L

Additional information

An acute Daphnia was not performed due to the toxicity seen and the low water solubility of the substance in the test media in a preliminary test. Therefore a long-term toxicity according to OECD TG 211 test was performed. 

Long-term Daphnia

An acute Daphnia was not performed due to the toxicity seen and the low water solubility of the substance in the test media in a preliminary test. Therefore a long-term toxicity according to OECD TG 211 test was performed. In accordance with OECD Guidance 23 (2000), water accommodated fractions (WAFs) of the substance were used. In a screening test toxicity was seen. No mortality was observed in the lowest WAF of 1 mg/l, whereas all daphnids exposed to WAFs prepared at 10 and 100 mg/L were found dead after 24 hours of exposure. Therefore, in the main test a WAF was prepared at 1.0 mg/L and stirred for 24 hours. Subsequently, the aqueous phase was siphoned off over glass wool to separate undissolved residue and used as the highest concentration. The lower test concentrations were prepared by diluting the highest concentration in test medium. This is in line with the OECD Guidance 23 where it says that at low concentrations dilutions should be used instead of preparing separate solutions of the testing concentrations. During preliminary testing it was observed that the Daphnia were sensitive about being transferred to new solutions. Based on further stability testing the substance was stable in test medium for 48 hours (69 -89% of initial). Therefore, test solutions in the main test were renewed every 48 hours.

Following a preliminary range-finding test in which toxicity was seen, in the main test parental Daphnia magna were exposed to solutions of the test material at nominal concentrations of 1.0, 3.2, 10, 32 and 100% of WAF prepared at a loading rate of 1.0 mg/L (one replicate and one parental Daphnia per concentration) for 21 days, under semi-static conditions. During the study samples for analyses were taken from the undiluted WAF at the beginning and the end of four intervals of 48 hours. Actual concentration measured in the freshly prepared 100% WAF ranged from 0.068 to 0.37 mg/L. Measured concentrations were at the level of 49 to 100% of initial at the end of the 48 h refreshment period. The calculated average exposure concentration was 0.17 mg/L (100% WAF). The lower exposure concentrations were calculated based on the dilution factor of 3.2 and corresponded to 0.0017, 0.0053, 0.017 and 0.053 mg/L in solutions containing 1.0, 3.2, 10, 32% WAF, respectively.

In the control, 15% mortality of adult animals was observed. Therefore, in test solutions mortality between 10 and 30% was considered not related to the applied dose.

In the control, 127 offspring were produced per surviving daphnid. At 0.0017, 0.0053, 0.017 and 0.053 mg/l (1, 3.2, 10 and 32% of the WAF) there were no effects on reproduction, growth and mortality. At 0.17 mg/l effects were seen in reproduction and growth: Reproduction decreased by 27.3% and growth with 13.5%. Mortality at this concentration (as well as at the highest concentration of 0.17 mg/l) was not affected, and the EC50 values were not reached > 0.17 mg/l.

A significant reduction of reproduction (as calculated per surviving daphnid) was observed in the highest group (27% reduction). There was no delay in the onset of reproduction. The body length of daphnids exposed to the highest concentration was significantly reduced at the end of the test (13% reduction). Under the conditions of the present study the test item did not affect reproduction and growth of Daphnia magna at 0.053 mg/L after 21 days of exposure (NOEC). Exposure to mean exposure concentrations of 0.17 mg/L induced significant inhibition of the reproductive capacity of the parental daphnids. The study met the acceptability criteria.