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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:
23 Mar - 29 Sep 2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Samples, in triplicate, were also taken on Days 20 (fresh media) and 21 (old media). The initial analysis of these samples failed due to high or low analytical quality control (QC) results. A set of the ‘back-up’ samples were analysed but again failed the analytical QC acceptance criteria. Given that the QC failures were generally at either the 0.2 and/or 150 µg/L levels, a further two QC levels at 0.5 and 50 µg/L were included in the analysis of the final set of ‘back-up’ samples. However, this batch again failed the QC acceptance criteria. The results from days 20 and 21 have therefore been presented for information only, to indicate that concentrations remained relatively consistent throughout the test, and have not been included in the calculation of the time-weighted mean measured concentrations.
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
adopted October 2012
Deviations:
yes
Remarks:
See section "Any other information on material and methods incl tables"
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples (8 mL) of test media from the control, solvent control and each test substance concentration were taken for analysis
- Sampling method: On Days 0, 1, 7, 14 and 20 samplles were taken from freshly prepared bulk test media. The corresponding old media Days 1, 2, 8, 15 and 21 were taken from pooled replicates. Triplicate samples were taken
- Sample storage conditions before analysis: Where analysis of ‘back-up’ samples was required, the samples were not stored beyond the proven frozen storage stability period of 8 days.
Vehicle:
yes
Remarks:
Dimethlyformamid
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: At the start of the test and at each media renewal, an initial solvent stock solution was prepared by dissolving ca 8 mg of test substance in a final volume of 10 mL of DMF to give a 0.8 mg/mL solvent stock solution. Serial dilutions were prepared in DMF to give further solvent stock solutions of 0.25, 0.0781, 0.0244 and 0.00763 mg/mL. An aliquot (100 µL) of the 0.00763, 0.0244, 0.0781, 0.25 and 0.8 mg/mL solvent stock solutions were separately spiked into 1000 mL of Elendt M4 medium to give the 0.763, 2.44, 7.81, 25 and 80 µg/L test concentrations, respectively.
Remark: During the range-finding test it was observed that the test item was not stable in the media following the same test item application as in the algae study (no usage of a solvent). The addition of a solvent stabilized the results without any effects on the Daphnia and do not bring the observed effects above the limit of water solubility. The detailed results of these solubility pre-test are summarized in an extra chapter within the study report.

- Controls: Yes , The control treatment was prepared with Elendt M4 medium only.
- Chemical name of vehicle: DMF (Dimethlyformamid)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 100 µL of DMF into 1000 mL of Elendt M4 medium (0.1 mL/L)
- Test concentration separation factor: 3.2
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water Flea
- Age at study initiation (mean and range, SD): < 24 h
- Method of breeding: The Daphnia magna are cultured in 1000 mL glass beakers containing 1000 mL of Elendt M4 medium. Each vessel and its contents are referred to as a 'culture'. New cultures are initiated with juvenile Daphnia magna (less than 24 hours old), at a density of 15-20 daphnids per vessel. The cultures are fed daily with a concentrated suspension of Chlorella vulgaris prepared in accordance with standard operating procedures.
- Source: Strain was originally obtained from MicroBio Tests Inc., Belgium
- Age of parental stock (mean and range, SD): Juveniles for use in toxicity tests were collected from the second brood onwards
- Feeding during test
- Food type: Suspension of Chlorella vulgaris
- Amount: 0.1 to 0.2 mg carbon/daphnid/day (typically between 1 and 5 x 10^E05 alga cells per mL, per day).
- Frequency: Fed daily

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: At the start of the test, a single juvenile Daphnia magna (<24 hours old) was randomly allocated to each test vessel and added using a wide bore pipette/glass tube. The internal bore of the pipette/glass tube was sufficiently wide to avoid damage of the animal during transfer.


Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
233 - 274 mg CaCO3/L
Test temperature:
19.0 - 20.1 °C
pH:
7.50 - 8.01 (Control)
7.72 - 8.13 (Solvent Control)
7.70 - 8.16 (Test item concentrations)
Dissolved oxygen:
98.2 - 103.6 % air saturation (Control)
91.4 - 103.1 % air saturation (Solvent Control)
84.4 - 104.2 % air saturation (Test item concentrations)
Conductivity:
621 - 701 µS/cm
Nominal and measured concentrations:
Control, Solvent Control, 0.763, 2.44, 7.81, 25 and 80 µg/L (nominal concentrations)
0,0, 0.273, 0.721, 1.87, 5.53 and 37 µg/L (based on time weighted average concentrations)
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: open
- Material, size, headspace, fill volume: Covered tall form glass beakers, 100 mL (nominal volume), containing ca 50 mL of test medium
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10
- Biomass loading rate: 5 mL per Daphnia

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Separate stock solutions of individual trace elements are first prepared in reverse-osmosis water. From these separate stock solutions, a single second stock solution is prepared which contains all trace elements as a combined solution.
- Alkalinity: 43 - 72 CaCO3/L
- Culture medium different from test medium: No, the same medium was used
- Intervals of water quality measurement: The pH, temperature and concentration of dissolved oxygen were determined in the freshly prepared test medium on Days 0, 7, 14 and 20. On Days 1, 8, 15 and 21 water quality measurements were determined using pooled replicate samples of test media at each of the nominal test concentrations.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16-hour light:8-hour dark cycle with an approximate 30 minute dawn/dusk transition period
- Light intensity: 1005, 1124 and 1008 lux at test start and 1011, 1136 and 1005 lux at test end

EFFECT PARAMETERS MEASURED: The adult Daphnia magna were observed for immobility, the presence or absence of eggs developing in the brood pouch (gravid or non-gravid) and mortality daily throughout the duration of the test. In addition, the number of juveniles present (alive or dead) were recorded, removed from the vessels and then discarded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Range finding study
- Test concentrations: 0.1, 0.5, 1.0, 10 and 100 µg/L
- Results used to determine the conditions for the definitive study: 100% mortality at 100 µg/L; mortality and reproduction results from the remaining test concentrations were similar to the control and solvent control groups
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1.87 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
Based on the total number of juveniles produced by surviving adults by Day 21. Juveniles produced by any daphnid which did not survive to Day 21 were not included in the analysis
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
5.53 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
Based on the total number of juveniles produced by surviving adults by Day 21. Juveniles produced by any daphnid which did not survive to Day 21 were not included in the analysis
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
2.64 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
Based on the total number of juveniles produced by surviving adults by Day 21. Juveniles produced by any daphnid which did not survive to Day 21 were not included in the analysis
Remarks on result:
other: 95% confidence limits
Remarks:
1.44 - 3.08 µg/L
Details on results:
- Observations on body length and weight: On comparison to the combined control groups, there were no statistically significant effects on the length of the adult daphnids surviving to Day 21 at any of the test substance concentrations remaining. The mean body length within the control and solvent conntrol was 3.5 mm
- Mortality of control: 10% parental mortality was observed in the control and 20% parental mortality in the solvent control
- Day of first brood: the first brood appeared after 9-10 days for the control, after 8-11 days for the solvent control, and after 8 -12 days for the nominal test item concentrations of 0.763, 2.44 and 7.81 µg/L; for the nominal test item concentration of 25 µg/L the first brood was observed at day 11-15.
Reported statistics and error estimates:
Statistical evaluation:
Statistical analysis was conducted on the data using the CETIS program v 1.8.6.8.
Data from the control and solvent control groups were compared using an Equal Variance t Two-Sample Test (reproduction and adult growth), Unequal Variance t Two-Sample Test (time to first brood) or a Fisher Exact Test (adult survival). The analysis showed no statistically significant differences between the control and solvent control groups therefore the results from the test group were analysed against the pooled control data.
To determine the No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC), the total number of juveniles produced by surviving adults by Day 21 and final adult lengths were analysed using the Dunnett Multiple Comparison Test. The day to first brood was analysed using the Dunn/Bonferroni-Holm Test and the number of adults surviving to Day 21 was analysed using the Cochran-Armitage Step-Down Test.

Chemical analysis:

Chemical analysis of the freshly prepared test media on Days 0, 1, 7 and 14 showed measured concentrations to range from 81% to 116% of nominal value with the exception of the 0.763 and 2.44 µg/L test concentrations on Day 0 were <80% of nominal. Given that these concentrations were below the No Observed Effect Concentration (NOEC), the results were not considered to affect the outcome of the test.

Analysis of the 24-hour old media on Days 1, 2, 8 and 15 showed measured concentrations to range from <LOQ to 23% of nominal value. The results from the media trial indicated that the test substance was stable in the test media over a 24-hour period. However, during the test, a significant decrease in measured concentrations was observed over each 24-hour period. This was considered to be possibly due to interaction of the test substance with the organic matter in the test vessels (test organisms, algal feed etc).

Samples, in triplicate, were also taken on Days 20 (fresh media) and 21 (old media). The initial analysis of these samples failed due to high or low analytical quality control (QC) results. A set of the ‘back-up’ samples were analysed but again failed the analytical QC acceptance criteria. Given that the QC failures were generally at either the 0.2 and/or 150 µg/L levels, a further two QC levels at 0.5 and 50 µg/L were included in the analysis of the final set of ‘back-up’ samples. However, this batch again failed the QC acceptance criteria. The results from days 20 and 21 have therefore been presented for information only, to indicate that concentrations remained relatively consistent throughout the test, and have not been included in the calculation of the time-weighted mean measured concentrations.

Given that a decline in measured concentrations was observed over each 24-hour period, the results are based on time-weighted mean measured test concentration. Where chemical analysis resulted in values of <LOQ, a value of half LOQ (0.1 µg/L) was used to calculate the time-weighted mean measured concentrations. The time-weighted mean measured concentrations were calculated to be 0.273, 0.721, 1.87, 5.53 and 37 µg/L, equivalent to 36, 30, 24, 22 and 46% of nominal, respectively.

Biological Results

 

The NOEC and LOEC values in terms of adult survival, day to first brood, juveniles produced per surviving parental Daphnia magna and adult growth, along with the ECx values for reproduction

Parameter

Toxicity value based on time-weighted mean measured concentrations (µg/L)

Adult survival*

Day to first brood

Juvenile production**

Growth

NOEC

5.53

5.53

1.87

5.53

LOEC

37

>5.53

5.53

>5.53

EC10

<0.273

-

2.64 (1.44 – 3.08)

-

EC20

5.87 (<0.273 – 8.29)

-

3.89 (2.68 – 4.84)

-

EC50

12.0 (4.89 – 14.8)

-

>5.53

-

* Based on observed mortality during the test
** Based on the total number of juveniles produced by surviving adults by Day 21. Juveniles produced by any daphnid which did not survive to Day 21 were not included in the analysis; - = Not applicable; Values in parentheses = 95% confidence limits

The mean number of live juveniles produced per treatment by Day 21, from surviving parental Daphnia magna

Nominal concentration
(µg/L)

Time-weighted mean measured concentration (µg/L)

Mean cumulative live juvenile production at Day 21

% Inhibition compared to the combined controls

Control

Control

64

0

Solvent control

Solvent control

88

0

0.763

0.273

85

-13

2.44

0.721

87

-16

7.81

1.87

81

-7

25

5.53

58

23

Validity criteria

Criterion from the guideline

Outcome

Validity criterion fulfilled

The mortality of the parent animals in the control (female Daphnia) does not exceed 20% at the end of the test.

10% parental mortality in the control

20% parental mortality in the solvent control

 

 

The mean number of living offspring produced per parent animal surviving at the end of the test is ≥ 60 in the control.

64 in the control

88 in the solvent control

Yes

Note: The same validity criterion (20%) can be used for accidental and inadvertent parental mortality for the controls as well as for each of the test concentrations.

 

Validity criteria fulfilled:
yes
Remarks:
See section:"Any other information on results incl. tables"

Description of key information

EC10 (21 d-reproduction): 2.64 µg/L (Daphnia magna, based on the time weighted average concentration)

Key value for chemical safety assessment

Additional information

One experimental study was available investigating the long-term toxicity of the substance to aquatic invertebrates. The reproduction test was conducted according to the OECD 211 (GLP) using the freshwater species Daphnia magna (Goodband, 2021). The Daphnids were exposed to the substance for 21 days in a semi-static design. The test solutions were renewed daily.

Due to the instability of the test item in the media, DMF was added. The nominal concentrations were performed with 0.763, 2.44, 7.81, 25 and 80 µg/L,based on the time-weighted average the test item concentrations were 0.273, 0.721, 1.87, 5.53 and 37 µg/L.

Chemical analysis of the freshly prepared test media on Days 0, 1, 7 and 14 showed measured concentrations to range from 81% to 116% of nominal value with the exception of the 0.763 and 2.44 µg/L test concentrations on Day 0 were <80% of nominal. Analysis of the 24-hour old media on Days 1, 2, 8 and 15 showed measured concentrations to range from <LOQ to 23% of nominal value. The results from the media trial indicated that the test substance was stable in the test media over a 24-hour period. However, during the test, a significant decrease in measured concentrations was observed over each 24-hour period. Given that a decline in measured concentrations was observed over each 24-hour period, the results are based on time-weighted mean measured test concentration.

The results from the test concentrations were compared to the combined controls because no difference were observed between control and solvent control. The EC10 for reproduction was calculated to be at 2.64 µg/L.

The validity criteria were satisfied, and therefore the results of the reproduction test are considered to be valid.

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