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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:
19 March - 17 July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
other: ISO International Standard - 10706
Version / remarks:
(2000)
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Solubility in water: <10 mg/L
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control) and 10 mg/L (loading rate)
- Sampling method: Samples for analysis were taken from all test concentrations and the control
- Frequency: At the beginning and at the end of three intervals of 48 hours (nominal days 0 and 2, 6 and 8, 12 and 14)
- Volume: 2.0 mL
- Sample storage conditions before analysis: samples were freshly analysed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A WAF was prepared at 10 mg/L by magnetic stirring for two days and subsequent centrifugation for 20 minutes at 10,000 rpm to remove undissolved test substance. The clear and colourless middle fractions were collected with a pipette from the centrifugation tubes and used for testing. Note that all glassware used (e.g. centrifugation tubes, test vessels and bottles used of the preparation) were pre-incubated for one day before start of the study to minimise possible loss of test substance due to adsorption to glassware.

- Controls: Test medium only (without test substance or other additives)

- Evidence of undissolved material (e.g. precipitate, surface film, etc): In the final test with a loading rate of 10 mg/L no undissolved material was reported.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: In-house laboratory culture with a known history
- Age at start of the test: < 24h old
- Feeding during test:
Twice daily, except on weekend days, 0.25 mL of Chlorella pyrenoidosa suspensions containing 0.44 to 0.69 x10^8 cells was added as feed for the daphnids. In the last week of exposure the daily ration was increased to 0.75 mL. This daily ration corresponded to at least 0.2 mg C/Daphnia/day, which is the recommended value for daily feeding per daphnid in the reproduction test according to the OECD Guideline 211.

ACCLIMATION
- Acclimation period: Not required

- Validity of the batch: 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.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
214 - 303 mg CaCO3/L
Test temperature:
19.1 - 20.5 °C
pH:
7.9 - 8.7
Dissolved oxygen:
8.1 - 10.6 mg/L
Nominal and measured concentrations:
- Nominal: A centrifuged solution prepared at a loading rate of 10 mg/L plus a control (0 mg/L)

- Measured:
Geometric mean concentration of test solution with daphnia and algal food: 3.5 mg/L
Geometric mean concentration of the abiotic control: 4.8 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: All-glass
- Type: Covered with Perspex plate
- Material, size, headspace, fill volume: All-glass, with 60 mL of capacity and filled with 50 mL of test solution.
All glassware (including centrifugation tubes) was pre-treated with the respective test solution for one day to minimise possible loss of test substance by adsorption to glass.
- Aeration: None
- Renewal rate of test solution (frequency): Every 48 hours
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 20
- No. of vessels per control (replicates): 20

TEST MEDIUM / WATER PARAMETERS
M7 medium was used.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light - 8 hours dark
- Light intensity:
At the start: 650-747 lux
At the end: 650-681 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Parental daphnids
Condition: Every workday, the number of living, immobile and dead parental daphnids was recorded. Dead daphnids were removed when observed.
Presence of eggs in the brood pouch: Every workday and upon renewal on non-workdays..
Body length: At the end of the test.

- Offspring
Appearance first brood: When observed.
Newborn daphnids: Every workday and upon renewal on non-workdays, the number of newborn young was counted and the condition of the young recorded. Thereafter the young were removed.
Presence of unhatched eggs: When observed.
Incidence of immobility: When observed.

RANGE-FINDING STUDY (7d)
- Test concentrations: A control and WAFs with the following loading rates 1.0, 10 and 100 mg/L. WAF solutions were prepared by 2d of magnetic stirring and 1 day of settlement. Subsequently, the middle fraction was siphoned for further testing..
- Results used to determine the conditions for the definitive study: No effects on survival or mobility of the parental daphnia were observed at any of the concentrations including a WAF prepared at 100 mg/L. Daphnids exposed to the WAF of 100 mg/L also produced the first eggs (on day 6), while most daphnids in all groups tested had produced eggs on day 7 of the test period.
Based on these observations it was concluded that Capcure 3-800 did not affect survival or development during the range-finding test up to and including a WAF prepared at 100 mg/l.
Analyses was performed on samples taken from the WAFs prepared at 1.0, 10 and 100 mg/L. Results showed that measured concentrations in freshly prepared WAFs were rather variable and generally ranged between 30 and 70% relative to the loading rates. Concentrations measured in the old solutions all decreased significantly during the renewal periods. The longer the renewal period, the higher the decrease. Furthermore, it was observed that decreases in the solutions incubated without daphnia and algae (abiotic solutions) were in general less significant. The latter indicated that food adsorption and/or metabolic processes may have contributed to the observed decrease.
Based on these results and the fact that the water solubility was indicated to be <10 mg/L (sponsor info) it was decided to continue with a semi-static limit test testing a WAF prepared at a loading rate of 10 mg/L. In consult with the sponsor it was further decided to pre-treat all glassware (including centrifugation tubes) with the respective test solution for one day to minimise possible loss of test substance by adsorption to glass. Removal of undissolved test substance was changed from a one-day settlement period to centrifugation (20’, 10,000 rpm).
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
3.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: parental mortality, reproduction and parental body length
Remarks on result:
other: Value determined by reviewer, based on concentration in test solutions with daphnia. Did not affect parental mortality, reproduction or parental body length up to solubility limit.
Details on results:
- Mortality of parent animals:
Control group: 2 (10% of total)
Loading rate 10 mg/L test group: 3 (15% of total)

- Cumulative mean number of living offspring produced per parent during the test period:
Control group: 94.0 ± 13.4 (SD)
Loading rate 10 mg/L test group: 92.6 ± 12.8 (SD)

- Average body length of parent animals (mm):
Control group: 4.19 ± 0.19 (SD)
Loading rate 10 mg/L test group: 4.27 ± 0.20 (SD)

- First time of presence of eggs in brood pouch: Day 5 (in both control and test group)
- First brood: At day 8 (in both control and test group)

- Other biological observations: There were no recordings of unhatched (aborted) eggs in the two test groups throughout the 21-day test period.
Reported statistics and error estimates:
Mortality:
The mortality among adult daphnids was biologically insignificant in the two groups tested, i.e. <20%. Therefore, no statistical analysis was performed.
Note that the OECD guideline allows for up to 20% control mortality for a test result to be valid. Consequently, any mortality rates ≤ 20% in a treatment group are considered incidental, and not substance-driven adverse effects.

Reproduction:
Total reproduction on day 21 was tested for normality and for homogeneity of variance and statistically tested using an ANOVA test followed by a mean comparison test (2 sample t-test; TOXSTAT Release 3.5, 1996).

Growth:
Since the mean body length in the test group at the end of the test was slightly higher than that in the control group and since this slight increase is not considered to be a substance-induced adverse effect, no statistical analysis was performed.
The respective threshold levels of effect (LOEC) and the respective NOECs were determined on basis of these statistics.

Table: Measured and mean exposure concentrations

Test group

Capcure 3-800

WAF (mg/L)

Mean exposure concentration (mg/L)*

Geometric mean study period

(mg/L)

Day 0-2

Day 6-8

Day 12-14

10

6.9-1.8 [3.5]

5.2-2.2 [3.4]

9.3-1.3 [3.5]

3.5

10 (abiotic)

6.9-4.6 [5.6]

5.2-3.2 [4.1]

9.3-2.5 [4.8]

4.8

* Results of fresh and spent analyses with geometric mean between brackets.

The initial concentrations measured at the start of the three analyses intervals were rather variable at the level of 52-93% of the applied initial loading rate. Similarly, as in the range-finding test, the measured concentrations were not stable during the refreshments. Further, as observed during the range-finding test, a slightly less pronounced decrease of concentrations was observed in absence of daphnids and algae (abiotic group). Hence, the decrease observed in the limit concentration was at least partly related to metabolisation and/or food absorption of test substance by the parental daphnids. The mean measured concentration calculated for the WAF prepared at 10 mg/Lwith daphnia and algal food was 3.5 mg/L and the mean in the abiotic group was calculated to correspond to 4.8 mg/L.

Validity criteria fulfilled:
yes
Remarks:
Mortality of parent animals in control < 20%; the cumulative of young per female in the controls was ≥60 (94±13)
Conclusions:
Under the conditions of the present study the test substance did not affect parental mortality, reproduction and parental body length of Daphnia magna at 3.5 mg/L after 21 days of exposure, being the geometric mean measured concentration prepared at a loading rate of 10 mg/L. Therefore, it was concluded that the test substance it is not toxic up to the solubility limit.
The 21-d NOEC to Daphnia magna is 3.5 mg/L
Executive summary:

In a 21-day semi-static reproduction study, the toxicity of the test substance to aquatic invertebrate Daphnia magna was assessed according to OECD Guideline No. 211 (2008) and GLP principles. Nominal concentration was prepared at a loading rate of 10 mg/L. The resulting geometric mean measured concentration was 4.8 mg/L in the test article control, containing test article without daphnia and 3.5 mg/L in the centrifuged test solutions with daphnia. The test substance did not affect parental mortality, reproduction and parental body length of Daphnia magna at the maximum solubility in test medium after 21 days of exposure. Therefore, it was concluded that the test substance it is not toxic up to the solubility limit. The author of the test report took the mean measured concentration in the test article control (4.8 mg/L) as a basis for the NOEC as he regarded the lower concentration in the test solutions at least partly related to metabolism by the parental daphnids

and/or food adsorption of the test substance. It is written in the report that it can be argued that the concentration in the test article control can be regarded as the bioavailable test substance concentration. The reviewer took the mean measured concentration in the centrifuged test solutions with daphnia (3.5 mg/L) as a basis for the NOEC because this is the actual mean measured concentration in the test solutions. Furthermore, there is no experimental evidence that the lower test concentration in the test solutions (compared to the test article control) is due to metabolism and/or food adsorption.

The 21-d NOEC to Daphnia magna is 3.5 mg/L.

All criteria for acceptability of the test were met according to OECD guideline No. 211 and the study is considered to be reliable without restrictions.

Description of key information

Under the conditions of the present study the test substance did not affect parental mortality, reproduction and parental body length of Daphnia magna at 3.5 mg/L after 21 days of exposure, being the geometric mean measured concentration prepared at a loading rate of 10 mg/L. Therefore, it was concluded that the test substance it is not toxic up to the solubility limit. 
The 21-d NOEC to Daphnia magna is 3.5 mg/L

Key value for chemical safety assessment

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

Additional information

In a 21-day semi-static reproduction study, the toxicity of the test substance to aquatic invertebrate Daphnia magna was assessed according to OECD Guideline No. 211 (2008) and GLP principles. Nominal concentration was prepared at a loading rate of 10 mg/L. The resulting geometric mean measured concentration was 4.8 mg/L in the test article control, containing test article without daphnia and 3.5 mg/L in the centrifuged test solutions with daphnia. The test substance did not affect parental mortality, reproduction and parental body length of Daphnia magna at the maximum solubility in test medium after 21 days of exposure. Therefore, it was concluded that the test substance it is not toxic up to the solubility limit. The author of the test report took the mean measured concentration in the test article control (4.8 mg/L) as a basis for the NOEC as he regarded the lower concentration in the test solutions at least partly related to metabolismby the parental daphnids

and/or food adsorption of the test substance. It is written in the report that it can be argued that the concentration in the test article control can be regarded as the bioavailable test substance concentration. The reviewer took the mean measured concentration in the centrifuged test solutions with daphnia (3.5 mg/L) as a basis for the NOEC because this is the actual mean measured concentration in the test solutions. Furthermore, there is no experimental evidence that the lower test concentration in the test solutions (compared to the test article control) is due to metabolism and/or food adsorption.

The 21-d NOEC to Daphnia magna is 3.5 mg/L.

All criteria for acceptability of the test were met according to OECD guideline No. 211 and the study is considered to be reliable without restrictions.