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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:
17 April 2018 - 20 November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
November 2018
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 1K72J
- Expiration date of the lot/batch: 02 October 2019
- Physical state/Appearance: Envigo - Light yellow powder
Sponsor - Light brown or light yellow powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and each surviving test group (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 6, 13 and 20 and of the expired test preparations on Days 1, 7, 14 and 21. Samples were stored frozen prior to analysis.
All sample bottles were fortified by the addition of 90 mL of acetonitrile in order to stabilize the test item.
Vehicle:
no
Details on test solutions:
An auxiliary solvent (Dimethylformamide, DMF) was used during a preliminary range-finding test, however prior to the definitive test it was determined that similar concentration levels could be achieved without the use of the auxiliary solvent, therefore the definitive test was performed without this solvent.

In the main test a nominal amount of test item (approximately 7.5 mg adjusted for test item purity) was dissolved in test water (Elendt M7 medium), with the aid of magnetic stirring for 20 minutes, and the volume adjusted to 20 liters to give a 0.36 mg/L stock solution. A series of dilutions were made from this stock solution to give test solutions of 0.0225, 0.045, 0.090 and 0.18 mg/L.
Each of the test solutions were inverted several times to ensure adequate mixing and homogeneity.

The test solutions were renewed daily throughout the test.

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia Magna
- Age at study initiation: Less than 24 hours old
- Method of breeding: Parthenogenesis
- Source: In-house laboratory cultures
- Feeding during test : mixture of algal suspension (Desmodesmus subspicatus) and GM300 flake food suspension, daily
- Amount: 5 to 20 µL algal suspension (Desmodesmus subspicatus) daily. 20 µL fish food suspension on days 0, 1, 2 and 3.
Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day dependent on the age and size of the animals. Equal amounts of food were given to each Daphnid.

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
238 - 264 mg/L as CaCO3
Test temperature:
20 - 21 °C
pH:
7.5 - 8.7 (control group)
7.7 - 8.9 (treatment groups)
Dissolved oxygen:
≥7.1 mg O2/L
Nominal and measured concentrations:
Nominal: 0.0225, 0.045, 0.090, 0.18, 0.36 mg/L
Measured concentrations used for assessment of endpoints: 0.0040, 0.0081, 0.014, 0.042, 0.071 mg/L. See "Any other information on materials and methods" for details on measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL glass vessels, 100 mL fill volume. Vessels were covered with a plastic lid to reduce evaporation
- Renewal rate of test solution: Daily
- No. of organisms per vessel: 1
- No. of vessels per concentration: 10 replicates
- No. of vessels per control: 10 replicates

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light / 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: Not exceeding 1500 lux

EFFECT PARAMETERS MEASURED:
The adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted using a stereo microscope before being discarded.
On a daily basis the numbers of live and dead of the "Parental" (P1) generation, the numbers of live and dead "Filial" (F1) daphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental daphnia as compared with the controls.
Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Daphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilization criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.
At the end of the test, the length of each surviving parent animal was determined.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0.0036, 0.036 and 0.36 mg/L
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.042 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.071 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.071 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks on result:
not determinable
Remarks:
Less than 50% immobilisation was seen at the highest concentration tested
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.004 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
(Body length)
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.008 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
(Body length)
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.071 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
(Body length)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.008 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.014 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.038 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% confidence interval: 0.033 - 0.043 mg/L
Details on results:
Lethal Effects on the Parental Generation (P1):
- Mortality: Mortalities were observed at the test concentrations of 0.014 and 0.071 mg a.i./L. Statistical analysis of the mortality data using the Fisher’s Exact Binomial Test with Bonferroni Correction showed that the observed mortalities in these two test groups were not significantly different (P≥0.05) when compared to the control group. However, it was considered that as 30% mortality was observed at 0.071 mg a.i./L compared to 0% in the control that the mortalities at 0.071 mg a.i./L should be considered as significant.

Sub-lethal Effects on the Parental Generation (P1):
- Behavioural abnormalities: None reported
- Observations on body length and weight: There were statistically significant differences between the control and the 0.0081, 0.014, 0.042 and 0.071 mg/L test groups in terms of the length of daphnids after 21 days exposure.
- Other biological observations: Pale daphnids were observed in the 0.0081, 0.014, 0.042 and 0.071 mg/L test groups with a clear dose relationship being apparent. The surviving daphnids in the 0.071 mg a.i./L test group also appeared to be small throughout the majority of the treatment period, one was also found to be coated with algae on one day only.
- Mortality of control: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No precipitation reported
- Effect concentrations exceeding solubility of substance in test medium: Not applicable

Effects on Reproduction:
- No statistically significant differences between the control and the 0.0040 and 0.0081 mg a.i./L test groups in terms of the number of live young produced per adult.
- The 0.014, 0.042 and 0.071 mg a.i./L test groups showed a statistically significant difference from the control after 21 days in terms of producing fewer numbers of live young per adult

Effects on the Filial Generation (F1):
- Information on the effects of the test item on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, all "filial" daphnids produced by the control and 0.0040 mg a.i./L test groups were observed to be of normal appearance, whereas, one brood of neonates in the 0.0081 mg a.i./L test group, six broods of neonates in the 0.014 mg a.i./L test group, twenty-seven broods of neonates in the 0.042 mg a.i./L test group and twenty-two broods of neonates in the 0.071 mg a.i./L were observed to be pale.

Analysis of the test solution showed that some test material found in the control group at a few occasions. Also, due to the decline in the measured concentration the results were based on the geometric mean measured test concentrations.
Reported statistics and error estimates:
The 21-Day ECx (immobilization) values and associated confidence limits were calculated by the Maximum-Likelihood Probit method.
The ECx (reproduction) values and associated confidence limits after 21 days were calculated by the Maximum-Likelihood Probit method.
The ECx (body length) values and associated confidence limits after 21 days were calculated by the 3-Parameter Normal Cumulative Distribution Function method.
All statistical analyses were performed using the ToxRat computer software package (ToxRat 3.2.1).
Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
Body length was the most sensitive endpoint, with a NOEC of 0.0040 and a LOEC of 0.0081 mg/L. The EC50 value was >0.071 mg/L. The NOEC for reproduction was 0.0081 mg/L.
Executive summary:

The long-term toxicity of the test item to aquatic invertebrates was studied in a Daphnia magna reproduction study according to EC Method C.20, OECD 211 and under GLP conditions.

10 neonates (<24 h old) were individually exposed in a semi-static sytem to nominal test concentrations of 0.0225, 0.045, 0.090, 0.18 and 0.36 mg/L for 21 days with daily renewal of test solutions. Additionally, a blank control was included with 10 neonates. Parental mortality, number of living and dead offspring, dicarded unhatched eggs were recorded and the lengths of the surviving parental daphnids were measured at the end of the test.

Given the decline in measured concentration it was considered appropriate to base the results on geometric mean measured test concentrations, in order to give a “worst case” analysis of the data. Corrected average geometric mean measured concentrations were used to assess the effect paramters and were: 0.0040, 0.0081, 0.014, 0.042 and 0.071 mg/L.

There was no mortality in the control group, mortality in the test item groups ranged from 10 to 30%. The 30% mortality was not statistically significantly different but should be considered significant. There was a statistically significant decrease in body length in all except the lowest test concentration. There was statistically significantly reduced reproduction in the three highest test concentrations.

Body length was the most sensitive endpoint in this study. The 21 -d NOEC for body length of Daphnia magna exposed to the test item was 0.0040 mg/L, the LOEC was 0.0081 mg/L. The 21 -d NOEC for reproduction was 0.0081 mg/L. The study met all validity criteria and is considered reliable without restrictions.

Description of key information

NOEC = 0.0040 mg/L (OECD 211, body length)

LOEC = 0.0081 mg/L (OECD 211, body length)

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.004 mg/L

Additional information

The long-term toxicity of the test item to aquatic invertebrates was studied in a Daphnia magna reproduction study according to EC Method C.20, OECD 211 and under GLP conditions.

10 neonates (<24 h old) were individually exposed in a semi-static sytem to nominal test concentrations of 0.0225, 0.045, 0.090, 0.18 and 0.36 mg/L for 21 days with daily renewal of test solutions. Additionally, a blank control was included with 10 neonates. Parental mortality, number of living and dead offspring, dicarded unhatched eggs were recorded and the lengths of the surviving parental daphnids were measured at the end of the test.

Given the decline in measured concentration it was considered appropriate to base the results on geometric mean measured test concentrations, in order to give a “worst case” analysis of the data. Corrected average geometric mean measured concentrations were used to assess the effect paramters and were: 0.0040, 0.0081, 0.014, 0.042 and 0.071 mg/L.

There was no mortality in the control group, mortality in the test item groups ranged from 10 to 30%. The 30% mortality was not statistically significantly different but should be considered significant. There was a statistically significant decrease in body length in all except the lowest test concentration. There was statistically significantly reduced reproduction in the three highest test concentrations.

Body length was the most sensitive endpoint in this study. The 21 -d NOEC for body length of Daphnia magna exposed to the test item was 0.0040 mg/L, the LOEC was 0.0081 mg/L. The 21 -d NOEC for reproduction was 0.0081 mg/L. The study met all validity criteria and is considered reliable without restrictions.