Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
October 2, 2012
Deviations:
no
Qualifier:
according to
Guideline:
other: HJ/T 153-2004, The guidelines for the testing of chemicals[S]. Beijing: SEPA, 2004
Deviations:
no
Qualifier:
according to
Guideline:
other: CRC-MEP. The Guidelines for the Testing of Chemicals, Effects on Biotic Sysmtem{M}. The 2nd edition. Beijing: China Enviroment Press. 2013:94-112
Deviations:
no
Qualifier:
according to
Guideline:
other: GB/T 21828-2008, Chemical-Daphnia magna reproduciton test[S]. Beijing: SAC:2008
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas
Details on test material:
- Purity: 99.9945%

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations Sampled: all control and test solutions were analysed
- Sampling Times: Samples were taken from all test solutions at the beginning (0 d), 3 d (aged), 9 d (fresh), 12 d (aged), 18 d (fresh), and 21 d (aged).
-Sampling method: 10.0 mL solution sample collected was drawn into 60.0 mL headspace bottles, the headspace bottles were placed in a water bath at 80±2°C for heating 30 minutes. Using a gas-tight syringe, 1.00 mL upper gas was drawn out from the headspace bottle, and manually injected into the GC to determine concentration.

Test solutions

Vehicle:
yes
Remarks:
Elendt M7
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION: Stock solution was prepared by adding the test water by ventilation for 6 hours from the cylinder into 1000 mL Elendt M7 medium, and the flow rate was about 4-7 bubbles per second. This stock solution was colourless and clear, and the concentration was analysed. The stock solution was then further diluted with Elendt M7 medium as necessary, to provide the remaining test concentrations. The test solutions were split charging to 50.0 mL Conical flask with cover (the full volume was about 72 mL). One control group was tested in parallel.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnid
- Strain/clone: Daphnia magna
- Source: Nanjing University
- Age of parental stock (mean and range, SD): colony was introduced in 2010; test species were 6 to 24 hours old at the start of the test
- Feeding during test
- Food type: Chlorella vulgaris Beij
- Amount: 0.1-0.2 mg C. vulgaris/Daphnia
- Frequency: once daily

ACCLIMATION
- Acclimation period: Daphnia magna, not being the first brood progeny, was the test species which were less than 24 hours old at the start of the test. All organisms used for a particular test originated from c
ultures established from the same healthy stock of Daphnia.
- Acclimation conditions (same as test or not): Yes

Study design

Test type:
semi-static
Water media type:
other: Elendt M7
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
TEST SYSTEM
- Test vessel: Conical flask with cover
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 50 mL (excluding headspace)
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): When the medium was renewed, a second series of test vessels were prepared and the parent animals were then transferred to them by a glass pipette of suitable diameter.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light and 8-hour dark
- Light intensity: 1000 to 1500 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): the number of living offspring was counted for each vessel daily. The offspring produced by each parent animal were preferably removed and counted daily from the appearance of the first brood to prevent them consuming food intended for the adult. Other parameters examined included time to production of first brood and number of broods per animal.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0, 10 and 100% (nominal)
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test, nominal concentrations of 0.15, 0.20, 0.25, 0.30 and 0.35 mg/L were conducted in the definitive test. Additionally, one blank control group was tested in parallel.

Test conditions

Hardness:
220-234 mg (CaC03)/L
Test temperature:
19.5-20.2°C
pH:
7.46-7.62
Dissolved oxygen:
7.92-8.78 mg/L
Nominal and measured concentrations:
Nominal: 0, 0.15, 0.20, 0.25, 0.30 and 0.35 mg/L
TWA analytical: 0, 0.047, 0.090, 0.186, 0.261, and 0.322 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flask with cover
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 50 mL (excluding headspace)
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): When the medium was renewed, a second series of test vessels were prepared and the parent animals were then transferred to them by a glass pipette of suitable diameter.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light and 8-hour dark
- Light intensity: 1000 to 1500 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): the number of living offspring was counted for each vessel daily. The offspring produced by each parent animal were preferably removed and counted daily from the appearance of the first brood to prevent them consuming food intended for the adult. Other parameters examined included time to production of first brood and number of broods per animal.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0, 10 and 100% (nominal)
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test, nominal concentrations of 0.15, 0.20, 0.25, 0.30 and 0.35 mg/L were conducted in the definitive test. Additionally, one blank control group was tested in parallel.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.261 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.322 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.186 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.261 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
During the test, all animals in the control were alive and the number of live offspring produced per parent animal surviving at the end of the test was 63~88. The coefficient of variation around the mean number of living offspring produced per parent animals in the control(s) was 10.3% (<25%). At the end of the test period, no adults died in blank control and 0.15, 0.20, and 0.25 mg/L treatments. 1 and 2 adults died in the 0.30 and 0.35 mg/L treatments, respectively. The reproduction rates in the treatments of 0.35 mg/L and the higher concentration treatments showed statistically significant difference from that of the control (p <0.05).
Reported statistics and error estimates:
The reproduction inhibition rate (Ir) for the individual animal was calculated. For the estimation of the lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC), the effects of the test substance on the mean reproduction for each concentration were compared with the control mean reproduction using a multiple comparison method of Dunnett's test.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
NOEC (Reproduction and Survival) = 0.186 mg/L (TWA concentration)
LOEC (Survival) = 0.261 mg/L (TWA concentration)
LOEC (Reproduction) = 0.322 mg/L (TWA concentration)
Executive summary:

Under semi-static conditions of 72-hour renewal, the effect on the reproductive output of Daphnia magna exposed to the test substance for 21 days was conducted according to "The guidelines for the testing of chemicals-effects on biotic systems" (HJ/T 153); "The Guidelines for the Testing of Chemicals"(the 2nd edition, 2013); Procedure 211 of the Daphnia magna, Reproduction Test", OECD Guidelines for Testing of Chemicals, adopted on October 2, 2012. During the test period, the pH values of the control and test media were between 7.46 and 7.62, the Dissolved Oxygen (DO) values varied from 7.92 to 8.78 mg/L at the test temperature, the hardness values varied from 220 to 234 mg (CaC03)/L, and the temperature of the control and test media was maintained in the range of 19.5°C to 20.2°C. All animals in the control were alive and the number of live offspring produced per parent animal surviving at the end of the test was 63-88. The coefficient of variation around the number of living offspring produced per parent animal in the control(s) was 10.3%. Therefore, the study met the acceptability criteria and was considered valid.

The concentration of the test substance in test medium was determined by GC-ECD to confirm the stability of the test substance in the test period. The analytical results showed that the concentration of the test substance was inconsistent in the test medium in several treatments throughout the test period (deviation over 20% of the initial concentration). Thus the test results were expressed with time weighted average concentration (TWA concentration). Under valid semi-static test conditions (72h-renewal), for Daphnia magna reproduction the LOEC was 0.322 mg/L (measured TWA concentration) and the NOEC was 0.261 mg/L (measured TWA concentration). For survival of adults, the LOEC was 0.261 mg/L (measured TWA concentration), the NOEC was 0.186 mg/L (measured TWA concentration).