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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:
From 09 October 2019 to 20 November 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: June 07th - June 10th, 2016 & July 13th, 2016 / Date of signature: January 03rd, 2017
Specific details on test material used for the study:
- Density (certified): 0,921 kg/dm3
- Water solubility: Insoluble
Analytical monitoring:
yes
Details on sampling:
At the start of the exposure renewal intervals, samples of the fresh media were taken after preparation of the WAFs and analyzed. At the end of the exposure renewal intervals (24 hours), samples of the 24-hours old media were taken from the test vessels. Samples were analyzed immediately (as far as technical possible) following sampling. For the sampling, at start of the first sampling interval, 40 g of the test solutions and from end of the first sampling interval on 50 g of the test solutions were directly weighted into the sampling vials.
- Preparation of the samples: 50 mL*) of the test item concentrations and the control were added with approx. 10% sodium chloride and liquid extracted with 2.5 mL cyclohexane by manual agitation for 2 min (enrichment factor 20). Before analysis, the organic phase was separated and dried with a small quantity of sodium sulfate. If necessary, to reach the calibration range, samples were further diluted with cyclohexane prior to analysis. 0.1 mL of internal standard solution (Undecanoic acid, derivatised, at 0.5 mg/L) were added to 1 mL of each sample prior to analysis.
- Sample storage: All samples were immediately extracted and the extracts were stored at room temperature. Until the start of the analysis the prepared samples were stored on an autosampler.
- Evaluation: Quantification of the test item was calculated by peak area based on the external standard, using the internal standard for correction purpose.

*) At start of the first sampling interval, 40mL of the test solutions were extracted with 2.0 mL cyclohexane.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Three water accommodated fractions (WAFs) at 100, 1000 and 10000 mg/L were separately prepared 120 ± 1 hour prior to the start of the exposure as well as 120 ± 1 hour prior to each renewal of the test solutions in an autoclaved glass flask (rinsed with cyclohexane and demineralized water) with a lateral outlet. For each loading rate, an appropriate amount of the test item (the density of the test item of 0.921 kg/dm3 was taken into account) was placed by pipette onto the surface of an appropriate amount of autoclaved dilution water. A slow stirring procedure was applied. Gentle stirring (to avoid formation of emulsion) was carried out for 120 ± 1 hour with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for 15 minutes at room temperature. Then, the WAF was removed by the lateral outlet of the glass flask from the approximate bottom of the water body. This procedure should not cause droplets by eye. The Tyndall test was not applicable as the dilution water itself caused a Tyndall effect. The mixing regime was selected based on the observations during the preliminary testing.
- Eluate: Culture Medium Elendt M4
- Differential loading: Three WAFs with nominal loading rates of 100, 1000 and 10000 mg/L of the test item were tested in an extended limit test.
- Controls: Dilution water without test item incubated under the same conditions as the test item groups. The control was treated equivalent to the preparation of the WAF mentioned above.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The WAF was checked microscopically for dispersed droplets (phase-contrast microscope, magnification: 400 times). No presence of dispersed droplets during the test was obsereved.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Age at study initiation (mean and range, SD): Less than 24 hours old daphnids from a healthy stock were used for the study. To achieve this juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period were used for the test. No first brood progeny was used for the test.
- Method of breeding: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 +/- 2 °C, in an incubator, 16 hours illumination; light intensity of max. 1500 lx
- Source: Daphnia magna STRAUS, obtained from continuous laboratory cultures. Origin = Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany.
- Culture feeding: The culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus,. The algae are cultured at the test facility.
- Feeding during test : Yes. Daily feeding with unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, 0.2 mg C / daphnia and day

ACCLIMATION
Acclimatization was not necessary, because the composition of the dilution water is equivalent to the culture medium
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
From 264 to 347 mg CaCO3/L.
The total hardness was above 140 mg/L as CaCO3.
Test temperature:
From 19.9 to 20.7°C.
The temperature was within the range of 20 ± 1 °C.
pH:
From 7.40 to 8.48.
Significant deviations ≥ 1.5 units between the initial and final pH-values were not observed.
Dissolved oxygen:
From 7.14 to 9.14 mg/L.
The dissolved oxygen concentration was above 3 mg/L.
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
- Nominal loading rates: 100, 1000, 10000 mg/L
- Measured concentrations: See table 6.1.1/8 in "Any other information on results incl. tables".
Details on test conditions:
TEST SYSTEM
- Test vessel: Headspace flasks, volume: 60 mL, loosely covered with transparent lids. For the start of exposure and each renewal brand-new test vessels were used
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Test volume 50 mL. 50 g test solution per replicate were weighed into the test vessels. This corresponds to 50 mL. The daphnids were transferred in a small amount of dilution water or test solution by pipette
- Aeration: Test vessels were not aerated during the test.
- Renewal rate of test solution (frequency/flow rate): The definitive test was performed under semi-static conditions with a daily renewal of the test solutions. For this purpose, a second set of test vessels were filled with the freshly prepared test solutions and the daphnids were transferred by pipette.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Same as the culture medium. Elendt M4, according to OECD 211, Annex 2.

OTHER TEST CONDITIONS
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Max. 1500 lx
- test temperature: The temperature was in the range of 18 – 22 °C and should not vary by more than 2°C.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality of parental daphnids: The number of parental daphnids, which were immobilized or dying during the test and the dates of death were observed and recorded once a day at least at the same time as the offspring was counted. Dead daphnids were removed as soon as they were detected.
- Neonates: First appearance of juveniles was checked daily. The number of neonates (alive and dead progeny) was counted and dates of release were recorded once a day from the first day of appearance of neonates until the end of the test. The neonates (alive and dead) were removed after counting and before addition of algae to prevent them from consuming food intended for the adults. The number of aborted eggs or dead offspring and dates of observation were recorded.
- Additional observations / condition of parental daphnids: Abnormalities (e.g. swimming behavior, number of males and winter eggs) were observed and recorded on each day of observation.
- Total body length / Mean dry weight of parental daphnids: At the end of the test, the total body length excluding the anal spine of each survived parental daphnid and the mean dry weight of the
survived parental daphnids of all loading rates and the control were determined. As stated in the OECD protocol, this may be useful additional information but is not used for determination of a NOEC.
- Additional endpoints: The time to first brood, the intrinsic rate of population increase and the number and size of first brood per animal were reported, but not used for endpoint calculations.

TEST CONCENTRATIONS
Based on the results of the non-GLP preliminary range finding test, the definitive reproduction test was carried out with three loading rates of the test item 100, 1000 and 10000 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
21 d
Dose descriptor:
EL10
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOELR
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
and adult mortality
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
>= 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
and adult mortality
Details on results:
- Reproductive output: The number of juveniles in all replicates of the control and the test groups was counted and recorded every day. The average number of living juveniles at the end of the test after 21 days per surviving parental daphnid was 77.1 in the control group and thus the validity criterion for the reproduction of a mean of ≥ 60 living offspring per surviving parent animal in the control was met. No parental daphnids were excluded due to accidental or inadvertent mortality from the evaluation of the reproductive output. The effects on reproduction were evaluated based on the reproduction
per survived parent animal. No significant trend in mortality was observed. Therefore, the evaluation has to be based on the cumulative offspring per survived parent. Further details are given in Table 6.1.4/1 in "Any other information on results incl. tables".

- Adult Mortality: No adult mortality was observed in the control and all nominal loading rates of the test item. No significant trend in mortality was observed. Accidental mortality as defined in the OECD test
guideline was not observed. See Table 6.1.4/2 in "Any other information on results incl. tables".

- Intrisic rates of natural increase (IR): The intrinsic rates of natural increase (IR) of the survived parental daphnids accounting for generation time and number of offspring were used for calculation of population growth and maintenance. For details, see Table 6.1.4/3 in "Any other information on results incl. tables".

- First appearance of living juveniles: The first appearance of living juveniles was observed on days 7 to 10 at all introduced parental daphnids of the control and the nominal loading rates. For details, see Table 6.1.4/4 in "Any other information on results incl. tables".

- Stillborn Juveniles and Aborted Eggs: In the control as well as in the loading rates, 100 and 1000 mg/L stillborn juveniles or aborted eggs were observed during the exposure period of 21 days. For details see Tables 6.1.4/5 and 6.1.4/6 4 in "Any other information on results incl. tables".

- Growth (Total Body Length and Mean Dry Weight) of the Survived Parental Daphnids: The mean values of the body length (excluding the anal spine) of the survived parental daphnids in the nominal loading rates of 100 to 10000 mg/L were 4.2 mm per daphnid and 4.1 mm per daphnid in the control. The mean dry weight of the survived parental daphnids was in the range of 0.43 to 0.45 mg per
daphnid in the nominal loading rates of 100 to 10000 mg/L and 0.40 mg per daphnid in the control (see Table 6.1.4/7 in "Any other information on results incl. tables").

- Presence of Males: No males were observed in the control or in the test groups during the test.

- Occurrence of Ephippia (Winter Eggs): No ephippia were observed in the control or in the test groups during the test.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 = 2.06 mg/L (95% CL: 1.00-4.00 mg/L)
- Valid range: 0.6 - 2.4 mg/L acc. to AQS P 9/2 (02/2000); clone 5, or 0.6 - 2.1 mg/L acc. to OECD 202 (2004); clone A.

Table 6.1.4/1: Effects on Adult mortality and Reproduction for Surviving Parents

(based on the nominal loading rates of the test item)

Nominal loading rate of the test item (mg/L)

Adult mortality (%)

Mean number of offspring per survived parental daphnid

Mean

SD

CV

10000

1000

100

Control

0

0

0

0

92.0

86.0

82.1

77.1

8.35

9.24

5.76

10.5

9.1

10.7

7.0

13.6

Table 6.1.4/2: Mortality (%) of the Adult Daphnids after 7, 14 and 21 days of exposure

(control: n = 10; test item loading rates: n = 10)

Nominal loading rate of the test item (mg/L)

Adult mortality (%)

7 days

14 days

21 days

10000

1000

100

Control

0

0

0

0

0

0

0

0

0

0

0

0

Table 6.1.4/3: Intrinsic rates of natural increase

Nominal loading rate of the test item (mg/L)

Mean IR

MV±SD

CV (%)

10000

1000

100

Control

0.374±0.0300

0.385±0.0489

0.356±0.0182

0.344±0.0169

8.0

12.7

5.1

4.9

Table 6.1.4/4: First appearance of living juveniles at the Survived Parental Daphnids in the Individual Groups

Nominal loading rate of the test item (mg/L)

Day of first appearance of living juveniles at the survived parental daphnids in replicate no.

First appearance mean day

1

2

3

4

5

6

7

8

9

10

10000

1000

100

Control

8

8

8

8

8

8

7

8

8

8

7

8

8

7

8

8

8

8

8

10

7

7

8

7

8

8

9

8

8

8

8

9

8

8

8

9

8

7

8

9

7.9

7.7

7.9

8.4

Table 6.1.4/5: Stillborn Juveniles and Aborted Eggs produced by the Survived Parental Daphnids after 21 Days

Nominal loading rate of the test item (mg/L)

Number of

Total no.

Number of introduced adults

Stillborn juveniles

Aborted eggs

Ʃ

N

10000

1000

100

Control

0

1

0

2

0

1

5

0

0

2

5

2

10

10

10

10

Table 6.1.4/6: Relative Number of Dead (Stillborn + Aborted Eggs) to Total Number of Juveniles per Survived Parental Daphnids

Nominal loading rate of the test item (mg/L)

Number of juveniles

Percentage of dead juveniles* (%)

Dead

Alive

Total

10000

1000

100

Control

0

2

5

2

920

860

821

771

920

862

826

773

0.00

0.23

0.61

0.26

Dead = aborted eggs + stillborn juveniles

Total = dead + alive juveniles

* related to the total number of juveniles

Table 6.1.4/7: Total Body Length and Dry Weight of the Survived Parental Daphnids

Nominal loading rate of the test item (mg/L)

Total length of the survived parent animals (mm)

Replicate no.

MV (mm)

N

Dry weight (mg)

1

2

3

4

5

6

7

8

9

10

Ʃ

MV

10000

1000

100

Control

4.0

4.0

4.0

4.0

4.0

4.5

4.5

4.5

4.5

4.0

4.5

4.0

4.0

4.5

4.0

4.0

4.5

4.0

4.0

4.0

4.0

4.0

4.5

4.0

4.5

4.0

4.0

4.5

4.5

4.0

4.0

4.0

4.0

4.5

4.5

4.0

4.0

4.0

4.0

4.0

4.2

4.2

4.2

4.1

10

10

10

10

4.3

4.4

4.5

4.0

0.43

0.44

0.45

0.40

N = number of the survived parental daphnids

Table 6.1.4/8: Measured concentrations of the test substance during the definitive test

First sampling interval

Sampling

Fresh medium

Day 5

Old medium

Day 6

Nominal loading rate of the test item (mg/L)

Calculated concentration* (µg/L)

Calculated concentration* (µg/L)

Group I

Group II

Group III

Sum**

Group I

Group II

Group III

Sum**

10000

1000

100

Control

12.2

59.9

2.80

<LOQ

25.2

123

5.79

<LOQ

11.0

55.3

2.81

<LOQ

48.5

239

11.4

<LOQ

0.573

0.174

0.287

<LOQ

1.09

0.325

0.449

<LOQ

0.523

0.137

0.185

<LOQ

2.18

0.635

0.921

<LOQ

Second sampling interval

Sampling

Fresh medium

Day 12

Old medium

Day 13

Nominal loading rate of the test item (mg/L)

Calculated concentration* (µg/L)

Calculated concentration* (µg/L)

Group I

Group II

Group III

Sum**

Group I

Group II

Group III

Sum**

10000

1000

100

Control

0.735

1.79

2.73

<LOQ

0.402

3.19

4.80

<LOQ

0.197

1.49

2.13

<LOQ

1.33

6.46

9.66

<LOQ

0.0974

0.0745

0.140

<LOQ

0.182

<LOQ

0.293

<LOQ

<LOQ

<LOQ

0.157

<LOQ

0.279

0.0745

0.590

<LOQ

Third sampling interval

Sampling

Fresh medium

Day 13

Old medium

Day 14

Nominal loading rate of the test item (mg/L)

Calculated concentration* (µg/L)

Calculated concentration* (µg/L)

Group I

Group II

Group III

Sum**

Group I

Group II

Group III

Sum**

10000

1000

100

Control

6.14

8.42

1.85

<LOQ

11.2

18.6

4.28

<LOQ

4.38

7.35

2.15

<LOQ

21.8

34.3

8.28

<LOQ

0.156

0.237

0.141

<LOQ

0.271

0.418

0.265

<LOQ

<LOQ

0.204

0.143

<LOQ

0.426

0.858

0.549

<LOQ

Forth sampling interval

Sampling

Fresh medium

Day 15

Old medium

Day 16

Nominal loading rate of the test item (mg/L)

Calculated concentration* (µg/L)

Calculated concentration* (µg/L)

Group I

Group II

Group III

Sum**

Group I

Group II

Group III

Sum**

10000

1000

100

Control

1.80

6.48

0.669

<LOQ

3.09

9.66

1.48

<LOQ

1.17

3.94

0.662

<LOQ

6.06

20.1

2.81

<LOQ

0.321

0.279

0.112

0.0383***

0.555

0.532

0.275

<LOQ

0.249

0.249

0.147

<LOQ

1.12

1.06

0.534

0.0383***

Fifth sampling interval

Sampling

Fresh medium

Day 19

Old medium

Day 20

Nominal loading rate of the test item (mg/L)

Calculated concentration* (µg/L)

Calculated concentration* (µg/L)

Group I

Group II

Group III

Sum**

Group I

Group II

Group III

Sum**

10000

1000

100

Control

0.462

0.328

1.11

<LOQ

0.375

0.616

2.19

<LOQ

0.183

0.341

1.07

<LOQ

1.02

1.28

4.37

<LOQ

0.286

0.0882

0.289

<LOQ

0.445

0.169

0.520

<LOQ

0.208

<LOQ

0.231

<LOQ

0.939

0.257

1.04

<LOQ

Sixth sampling interval

Sampling

Fresh medium

Day 20

Old medium

Day 21

Nominal loading rate of the test item (mg/L)

Calculated concentration* (µg/L)

Calculated concentration* (µg/L)

Group I

Group II

Group III

Sum**

Group I

Group II

Group III

Sum**

10000

1000

100

Control

6.71

2.21

0.0844

<LOQ

14.3

13.5

<LOQ

<LOQ

5.57

9.93

<LOQ

<LOQ

26.6

25.7

0.0844

<LOQ

0.139

0.110

0.0249

<LOQ

0.279

0.822

<LOQ

<LOQ

<LOQ

0.606

<LOQ

<LOQ

0.418

1.54

0.0249

<LOQ

* = Group I (Peak 1 to 3); group II (Peak 4 to 6); group III (Peak 7 to 9). Calculated concentration taking enrichment, dilution factor and the percentage of each group into account.

** = Sum of group I to III, only measured values > LOQ taken into account

*** = Control group I > LOQ due to a slight contamination for unknown reasons

LOQ = Limit of quantification, group I: 0.1 µg test item/L, corresponding to 0.024 µg a.i./L; group II: 0.3 µg test item/L, corresponding to 0.14 µg a.i./L, group III: 0.6 µg/L test item/L, corresponding to 0.13 µg a.i./L.

Validity criteria fulfilled:
yes
Conclusions:
The effects on reproduction were evaluated based on the reproduction per survived parent animal. No significant trend in mortality was observed. Therefore, the evaluation has to be based on the cumulative offspring per survived parent. The overall effect threshold for effects of the test item under the test conditions was ≥ 10000 mg/L (NOEC) and > 10000 mg/L (LOEC, EL10) based on the nominal loading rates of the test item.
Executive summary:

A Daphnia magna reproduction test (semi-static, 21 d) of the test substance was conducted at the test facility according to OECD Guideline 211 (2012) from 2019 -10 -25 to 2019 -11 -20 with the definitve exposure phase from 2019 -10 -31 to 2019 -11 -20. Test species was Daphnia magna STRAUS. Ten daphnids, held individually, were used per loading rate and control. At test start the daphnids were 2 to 24 hours old. The test item is a UVCB substance. Therefore, three water accommodated fractions (WAF) with a nominal loading rate of 100 – 1000 – 10000 mg/L were tested under semi-static conditions with a daily renewal of the test solutions. Aim of the test was to assess the effects on the reproduction capacity and other sub-lethal effects. The nominal loading rates of the test substance were selected based on the results of a (non-GLP) shortened reproduction test (until observation of the first brood) under semi-static conditions with daily water renewal over a period of 9 days. The concentrations of the test substance were analytically verified via GC-MS in fresh media on days 5, 12, 13, 15, 19 (0 hours) and in old media on days 6, 13, 14, 16, 20 (24 hours) in all nominal loading rates and the control. As the active ingredient of the test item shows 9 significant peaks, a group evaluation of three major groups took place: group I with peaks 1 to 3, group II with peaks 4 to 6 and group III with peaks 7 to 9. For the evaluation all effects levels were based on the nominal loding rates of the test substance. The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were met.

The effects on reproduction were evaluated based on the reproduction per survived parent animal. No significant trend in mortality was observed. Therefore, the evaluation has to be based on the cumulative offspring per survived parent. The overall effect threshold for effects of the test item under the test conditions was ≥ 10000 mg/L (NOEC) and > 10000 mg/L (LOEC, EL10) based on the nominal loading rates of the test item.

Description of key information

OECD Guideline 211, GLP, key study, validity 1:

21d-EL10 reproduction (Daphnia magna) > 10000 mg/L based on nominal loading rates.

Key value for chemical safety assessment

Additional information

One key study is available to assess the long-term toxicity of the registered substance to aquatic invertebrates.

In this reproduction test with Daphnia magna (NOACK, 2020), the effects on the reproduction capacity and other sub-lethal effects of three water accommodated fractions (WAF) with nominal loading rates of 100 – 1000 – 10000 mg/L of the registered substance were determined at the test facility according to OECD Guideline 211 with GLP compliance. This study was performed over a period of 21 days under semi-static and closed conditions with a daily renewal of the test solutions. The concentrations of the test substance were analytically verified via GC-MS in fresh media on days 5, 12, 13, 15, 19 (0 hours) and in old media on days 6, 13, 14, 16, 20 (24 hours) in all nominal loading rates and the control. The effects on reproduction were evaluated based on the reproduction per survived parent animal. No significant trend in mortality was observed. Therefore, the evaluation has to be based on the cumulative offspring per survived parent. The overall effect threshold for effects of the test item under the test conditions was ≥ 10000 mg/L (NOEC) and > 10000 mg/L (LOEC, EL10) based on the nominal loading rates of the test substance.

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