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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Study initiation and experimental starting date: January 4, 2017; Experimental completion date: February 17, 2017, Study completion date: June 29, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, effects on Biotic Systems, 202 Acute Immobilization Test. [M]. Second edition. Beijing: China Environmental Science Press. 2013: 22~29
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: China National Technical Committee for Standardization of Dangerous Chemicals Management. GB/T 21830-2008 Chemicals-Daphnia sp., acute Immobilization Test [S]. Beijing: Standards Press of China, 2008.
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
Version / remarks:
15 October, 2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: State Environmental Protection Administration of China. The Guidelines for the test of chemical (HJ/T 153-2004). Beijing: China Environmental Science Press.2004.
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Sample pretreatment and preservation
Samples of the 100 mg/L test solution were filtered through a 0.45 μm filter membrane (discarded the first 5-10 mL filtrations), then the filtration of was diluted for 10-folds with deionized water before analysis. Blank control was filtered through a 0.45 μm filter membrane before analysis. The samples were analyzed at the same day.
Vehicle:
yes
Remarks:
Elendt M4 medium was used in the test. The test medium was prepared with deionized water.
Details on test solutions:
0.100 g test substance was obtained in a small glass-beaker and dissolved with a small amount of Elendt M4 medium. Then the mixture was transferred into a 1000 mL triangular bottle and diluted with test medium to 1000 mL, the nominal concentration of 100 mg/L test solution was obtained and used in the test directly.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Daphnia magna
- Justification for species other than prescribed by test guideline: not applicable
- Source: The stock animals were introduced from the Safety Evaluation Center, Shenyang Research Institute of Chemical Industry (introduced batch No. was DM20130801-1). Daphnia magna used in this study were cultured in our laboratory, and the acclimated batch No. of test animal was DMU20161222-1.
- Age of parental stock (mean and range, SD):
- Feeding during test: The daphnids were not fed during the exposure.
- Food type: concentrated algal suspension
- Amount: not available
- Frequency: not available
The animals were less than 24 hours old and were not first brood progeny.

ACCLIMATION
- Acclimation period: 48 h
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: concentrated algal suspension
- Feeding frequency: not available
- Health during acclimation (any mortality observed): no

At the start of the test, Daphnia magna were acclimated in the testing laboratory for 48h at least under the same conditions of the study (test medium: Elendt M4 medium, temperature: 20 °C ± 2 °C, light and dark period: 16 h light / 8 h dark, illumination intensity: 1000 lx -1500 lx). During the acclimation, Daphnia magna were fed with a concentrated algal suspension. (See the Daphnia magna acclimation record of DMU20161222-1).
The number of test daphnia: 40 animals in total
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
The total hardness of test medium was 240 mg/L (expressed as CaCO3), and TOC (Total organic carbon) concentration was 0.748 mg/L.
Test temperature:
19.8 °C - 20.8 °C, the total variability was < 2 °C during the whole test.
pH:
The test was performed without adjustment of pH. The pH of the test medium ranged from 7.82 to 7.84 and the variation was less than 1.5 units during the test.
Dissolved oxygen:
The test solutions were not aerated during the exposure. The dissolved oxygen concentration in the test solutions during the test ranged from 8.3 mg/L to 8.4 mg/L, it was kept above 3 mg/L.
Salinity:
not specified
Conductivity:
not specified
Nominal and measured concentrations:
According to the test substance information provided by the sponsor, the “Acute Aquatic Toxicity Data” showed that the 48h-EC50 for the Daphnia acute immobilization was greater than 100 mg/L, so a limit test was performed with a nominal concentration of 100 mg/L test group.
Details on test conditions:
TEST SYSTEM
According to the stability test of the fish, juvenile growth test (Study number: 2016ESG0207), the measured concentration of test substance was stable during 4 days. Consequently, the acute immobilization test of test substance with Daphnia magna was performed under static condition.

- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Volume of test solution: 50 mL / test vessel
- Aeration: No aeration
- Type of flow-through (e.g. peristaltic or proportional diluter): no flow-through, static test conditions
- Renewal rate of test solution (frequency/flow rate): no
- No. of organisms per vessel: 5 daphnids / test vessel, 40 daphnids in total
- No. of vessels per concentration (replicates): 4 replicates / test group
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): -
- Biomass loading rate: not available

TEST MEDIUM / WATER PARAMETERS
Elendt M4 medium was used in the test. The test medium was prepared with deionized water (see Annex A). The total hardness of test medium was 240 mg/L (expressed as CaCO3), and TOC (Total organic carbon) concentration was 0.748 mg/L.
Separate stock solutions (I) of individual trace elements are first prepared in water of suitable purity, e.g. deionised, distilled or reverse osmosis. From these different stock solutions (I) a second single stock solution (II) is prepared, which contains all trace elements (combined solution).

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:
- Light intensity: Artificial light of white fluorescent lamp was used in the test, and the photoperiod was 16 h light / 8h darkness.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
not performed
Reference substance (positive control):
yes
Remarks:
The sensitivity test of reference substance K2Cr2O7 showed that, the 24h - EC50 of K2Cr2O7 to D. magna was 1.21 mg/L (95% confidence limits in the range of 1.13 mg/L to 1.29 mg/L), so within the required range of 0.6 mg/L to 2.1 mg/L for validity.
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Immobilities of each group at 24 h and 48 h after the start the test were calculated. The 24 h and 48 h - EC50 values of the test substance to Daphnia magna were calculated by Binomial Distribution method (CT-TOX Multi-Method Program, IBM PC Version 1.1).
Results with reference substance (positive control):
The sensitivity test of reference substance K2Cr2O7 to Daphnia magna showed that, the 24 h - EC50 of K2Cr2O7 to Daphnia magna was 1.21 mg/L and the 95% confidence limits was in the range of 1.13 mg/L to 1.29 mg/L. The 24 h - EC50 was within the range of 0.6 mg/L to 2.1 mg/L, which met the requirement for the validity.

Environmental conditions

Intensity of illumination at the start and end of the test was 1144 lx and 1129 lx on average, respectively. During the test, the continuously monitoring of the water temperature was in the range from 19.8 °C to 20.8 °C.

During the test, the measured pH value was in the range of 7.82 to 7.84 and the dissolved oxygen was in the range of 8.3 mg/L to 8.4 mg/L.

Measurement of test substance concentrations in test solution

(A) Samples of the 100 mg/L test solution were filtered through a 0.45 μm filter membrane (discarded the first 5-10mL filtrations), then the filtration of was diluted for 10-folds with deionized water before analysis. Blank control was filtered through a 0.45 μm filter membrane before analysis. The samples were analyzed at the same day.

(B) Measured concentrations of test substance in test solutions

The results of the analytical methods validation are presented in the final report of “Validation of An Analytical Method of Tamisolve NxG” (The report No. is A16074-T16064-1R). The limit of detection (LOD) was 0.0282 mg/L and limit of quantitation (LOQ) was 0.0942 mg/L.

At the start (0 h) and end (48 h) of the test, the measured concentrations of the blank control were both lower than LOD (0.0282 mg/L).

At the start (0 h) and end (48 h) of the test, the measured concentrations of 100mg/L test substance group were 99.2 mg/L and 97.8 mg/L, respectively, and equaled to 99% and 98% of nominal concentration 100 mg/L.

(C) Immobility

At 24 h and 48 h after the start of the test, the immobilities of the blank control for Daphnia magna were both 0%, which were below 10% and met the requirement.

At 24 h and 48 h after the start of the test, the immobilities of the nominal concentration of 100 mg/L test solution for Daphnia magna were both 0%.

(D) Median effective concentration (EC50)

At the end of the test, the 48 h median effective concentration (EC50) of test substance to Daphnia magna was greater than the nominal concentration of 100 mg/L.

Table 1 Immobilities of daphnids in the test

Test group

Replicate

Number of daphnids

24 h

48 h

Immobilized number

Immobility (%)

Mean Immobility (%)

Immobilized number

Immobility (%)

Mean Immobility (%)

Blank control

1

5

0

0

0

0

0

0

2

5

0

0

0

0

3

5

0

0

0

0

4

5

0

0

0

0

100mg/L group

1

5

0

0

0

0

0

0

2

5

0

0

0

0

3

5

0

0

0

0

4

5

0

0

 

0

0

Table 2 Measured concentrations of test substance in test solutions

Test group

Measured concentrations of test substance (mg/L) (The percent of measured concentration versus the nominal concentration)

0 h

48 h

Blank control

<LOD

<LOD

100mg/L group

99.2

97.8

(99%)

(98%)

Note: The limit of detection (LOD) was 0.0282mg/L and the limit of quantitation (LOQ) was 0.0942mg/L.

Validity criteria fulfilled:
yes
Remarks:
Immobilites of the blank control were 0%, thereby beeing always below 10%. The dissolved oxygen concentration of test solutions ranged from 8.3-8.4 mg/L, thereby always above 3 mg/L. The 24h - EC50 of K2Cr2O7 was within the range of 0.6 mg/L to 2.1 mg/L.
Conclusions:
According to the test substance information provided by the sponsor, the “Acute Aquatic Toxicity Data” showed that the 48h-EC50 for the Daphnia acute immobilization was greater than 100 mg/L, a limit test was performed with a nominal concentration of 100 mg/L test group and a blank control containing no test substance.
Executive summary:

In order to evaluate the possible acute toxic effects on the mobility to Daphnia magna for a period of 48 hour, the acute toxicity of N-Butylpyrrolidone towards Daphnia magna was investigated in a study according to OECD Guideline No 202. According to the test substance information provided by the sponsor, the “Acute Aquatic Toxicity Data” showed that the 48h-EC50 for the Daphnia acute immobilization was greater than 100 mg/L, a limit test was performed with a nominal concentration of 100 mg/L test group and a blank control containing no test substance. 0.100 g test substance was obtained in a small glass-beaker and dissolved with a small amount of Elendt M4 medium. Then the mixture was transferred into a 1000 mL triangular bottle and diluted with test medium to 1000 mL, the nominal concentration of 100 mg/L test solution was obtained and used in the test directly. At the beginning and at the end of the test, the actual content of the test item in the test solutions and the blank control were measured by RRLC at the start (0 h) and end (48 h) of the test.

Four test vessels were filled with 50 mL volumes of test medium or 100 mg/L test substance solution in the 150 mL glass-beakers as the blank control groups or test substance groups, then young daphnids (5/vessel), aged less than 24 h at the start of the test, were exposed to 100 mg/L of the test substance for a period of 48 hours (they were placed into each test vessel to start the test) . Immobilisation was recorded at 24 h and 48 h and compared with control values. The results were analyzed in order to calculate the EC50 at 48 h.

Under the test condition of this study, acute immobilization test of test substance with Daphnia magna was performed as limit test under static test conditions. At the end of the test, the immobility of the nominal concentration of 100 mg/L test solution to Daphnia magna was 0%. The treatment showed no toxicity towards daphnids. None of the animals was immobilised in the control.

Hence, the results for the 48 h median effective concentration (EC50), the highest concentration causing no immobility (EC0) and the lowest concentration producing 100 percent immobility (EC100) of test substance for Daphnia magna would be described based on nominal concentration.

EC50 > 100 mg/L (nominal concentration)

EC0 = 100 mg/L (nominal concentration)

EC100 > 100 mg/L (nominal concentration)

Description of key information

The acute toxicity of N-Butylpyrrolidone towards Daphnia magna was investigated in a key study according to OECD Guideline No 202 and EU method C.2. At the beginning and at the end of the test, the content of the test item in the test solutions was determined using GC-determination. The main study was performed as limit test using 100 mg/L nominal concentration. Twenty daphnids were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted. The recovery after 48 hours was 94 % of the start concentration, the correlation between nominal and measured concentration was good. Therefore, the determination of the biological results was based on the nominal concentration. The following results were determined for the test item N-Butylpyrrolidone (species: Daphnia magna). 24h-NOEC >100 mg/L, 48h-NOEC >100 mg/L, 24h-EC50i > 100 mg/L, 48h-EC50i > 100 mg/L. The treatment showed no toxicity towards daphnids. None of the animals was immobilised in the control.

In a supporting study the possible acute toxic effects on the mobility to Daphnia magna was evaluated for a period of 48 hour. The acute toxicity of N-Butylpyrrolidone towards Daphnia magna was investigated in a study according to OECD Guideline No 202. According to the test substance information provided by the sponsor, the “Acute Aquatic Toxicity Data” showed that the 48h-EC50 for the Daphnia acute immobilization was greater than 100mg/L, a limit test was performed with a nominal concentration of 100 mg/L test group and a blank control containing no test substance. 0.100 g test substance was obtained in a small glass-beaker and dissolved with a small amount of Elendt M4 medium. Then the mixture was transferred into a 1000mL triangular bottle and diluted with test medium to 1000mL, the nominal concentration of 100mg/L test solution was obtained and used in the test directly. At the beginning and at the end of the test, the actual content of the test item in the test solutions and the blank control were measured by RRLC at the start (0h) and end (48h) of the test.

Four test vessels were filled with 50 mL volumes of test medium or 100 mg/L test substance solution in the 150 mL glass-beakers as the blank control groups or test substance groups, then young daphnids (5/vessel), aged less than 24h at the start of the test, were exposed to 100 mg/L of the test substance for a period of 48 hours (they were placed into each test vessel to start the test) . Immobilisation was recorded at 24h and 48h and compared with control values. The results were analyzed in order to calculate the EC50 at 48h.

Under the test conditions of this study, acute immobilization test of test substance with Daphnia magna was performed as limit test which conducted under static test. At the end of the test, the immobility of the nominal concentration of 100 mg/L test solution to Daphnia magna was 0%. The treatment showed no toxicity towards daphnids. None of the animals was immobilised in the control.

Hence, the results for the 48 h median effective concentration (EC50), the highest concentration causing no immobility (EC0) and the lowest concentration producing 100 percent immobility (EC100) of test substance for Daphnia magna would be described based on nominal concentration: EC50 and EC100 were found to be > 100 mg/L (nominal concentration), the EC0 is equal to 100 mg/L (nominal concentration).

In conclusion, both studies revealed consistent results, which do not require classification or labelling as toxic to aquatic invertebrates.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information