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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-07-26 to 2019-08-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: June 07th - June 10th, 2016 & July 13th, 2016 / Date of signature: January 3rd 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 and at the renewal (0 and 24 hours), samples were taken after preparation of the WAFs and analyzed. At the renewal and at the end of the exposure (24 and 48 hours),
samples of the 24-hours old media were taken from additional replicates (since volumes of the test replicates are insufficient for the analytical determination of the test item concentration), which were prepared for the sampling of the old media with test media, but without daphnids. These replicates were incubated under test conditions until sampling. For the sampling, 50.0 ± 0.1 g of the test solutions were directly weighted into the sampling vials.
- Preparation of the samples: 50 mL of the test item concentrations, the solvent control 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 at test start were further diluted factor 10 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.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Three water accommodated fractions (WAF) 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 the renewal of the test solutions in an autoclaved glass flask (rinsed with cyclohexane and demineralized water) with a lateral outlet. The procedure according to ASTM D6081 (2014) was carried out. Since microbial growth was observed in the 100 mg/L approach for the renewal of the test solutions on day 1, the approach which was used for the start of exposure was stirred one day longer and was used for the renewal on the next day instead. 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 (approx. 144 hours for the WAF 100 mg/L used at the renewal of the test solutions) 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 column. The mixing regime was selected based on the observations during the preliminary testing.
- Eluate: autoclaved dilution water
- Differential loading: 100, 1000 and 10000 mg/L
- Controls: Autoclaved dilution water without test item incubated under the same conditions as the test groups. The control was treated equivalent to the preparation of the WAF mentioned above. Since microbial growth was observed in the control approach for the renewal of the test solutions on day 1, the approach which was used for the start of exposure was stirred one day longer and was used for the renewal on the next day instead.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): This procedure caused did not cause droplets by eye. The Tyndall test is not applicable as the dilution water itself caused a Tyndall effect. The WAFs were checked microscopically for dispersed droplets (phase-contrast microscope, magnification: 400 times). No dispersed droplets were observed in any of the tested WAFs.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Daphnia magna STRAUS
- Reason for the selection of the test system: Daphnia magna is the preferred species in accordance with the test guidelines and is bred at the test facility.
- Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
- Breeder: Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- 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.
- Feeding during test : The daphnids were not fed during the study.
- Food type: 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.

ACCLIMATION
Acclimatization of the daphnids was not necessary, because the composition of the dilution water was equivalent to the culture medium.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
none
Post exposure observation period:
none
Hardness:
Total hardness from 242 to 244 mg CaCO3/L
Test temperature:
18 - 22°C, constant within ± 1°C
pH:
From 7.51 to 8.58
Dissolved oxygen:
From 7.11 to 8.68 mg Dissolved O2/L
Salinity:
Not applicable
Conductivity:
From 570 to 618 µS/cm
Nominal and measured concentrations:
- Nominal loading rate: 100 - 1000 - 10000 mg/L
- Measured concentrations: Details of the analytical results are presented in Table 6.1.3/2 in "Any other information on results incl. tables"
Details on test conditions:
TEST SYSTEM
- Test vessel: Headspace flasks, volume: 50 mL, loosely covered with watch glasses
- Type (delete if not applicable): closed
- Renewal rate of test solution (frequency/flow rate): The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Application: 20 g test solution per replicate were weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by pipette.

TEST MEDIUM / WATER PARAMETERS
- Culture medium: Elendt M4, according to OECD 202, Annex 3 (2004) was used.

OTHER TEST CONDITIONS
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 1500 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobilization was determined in all groups after 24 and 48 hours. An animal was considered immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Observation of other adverse, sub-lethal effects (e.g. discoloration, abnormal behavior etc.) was done, but none occurred during the course of the study.

RANGE-FINDING STUDY
A non-GLP preliminary range finding test under semi-static conditions over a period of 48 hours was conducted at the test facility with three WAFs with nominal loading rates of the test item of 100, 1000 and 10000 mg/L. The WAFs were individually prepared five days prior to the start of the exposure (day -5) and five days prior to the renewal of the test solutions (day -4). The test item was analytically verified via GC-MS/MS in the WAF and in the control in the fresh media at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) as well as in 24-hours old media at the renewal and at the end of the exposure (24 and 48 hours). The preliminary range finding test was conducted under diffuse light conditions ( light intensity of max. 1500 lx, 16/8 hours light/dark cycle). In the range finding test, two replicates per concentration level and control, each with five daphnids, were tested.
No effects on Daphnia magna were observed in any of the tested WAFs.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9)
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other:
Remarks:
The ELx-values are based on the nominal loading rates of the test item, which is recommended by OECD guidance document No. 23 (2019) for testing of WAFs.
Details on results:
- No effects on Daphnia magna were observed in any of the tested WAFs, see details in Table 6.1.3/1 in "Any other information on results incl. tables"
- The WAFs were colorless and clear. No undissolved test item was present in any of the tested WAFs. This was checked microscopically (phase-contrast microscope, magnification: 400 times).
Results with reference substance (positive control):
The percentage of immobility for the reference item potassium dichromate was determined after 24 hours from 2019-08-07 to 2019-08-08.
24h-EC50 = 2.00 mg/L (confidence limits: 1.00 - 4.00 mg/L)
(valid range 0.6 - 2.4 mg/L according to AQS P 9/2 (02/2000))
Reported statistics and error estimates:
Since no effects on Daphnia magna were observed in any of the tested WAFs, no calculations were made.

Table 6.1.3/1: Absolute numbers of immobile Daphnids after 24 and 48 hours of exposure in the definitive test

(n=20, divided into 4 replicates with 5 daphnids each)

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

NUMBER OF IMMOBILE DAPHNIDS / TOTAL NUMBER OF DAPHNIDS

24 hours

48 hours

Replicates

Replicates

1

2

3

4

Sum

1

2

3

4

Sum

10000

0 / 5

0 / 5

0 / 5

0 / 5

0 / 20

0 / 5

0 / 5

0 / 5

0 / 5

0 / 20

1000

0 / 5

0 / 5

0 / 5

0 / 5

0 / 20

0 / 5

0 / 5

0 / 5

0 / 5

0 / 20

100

0 / 5

0 / 5

0 / 5

0 / 5

0 / 20

0 / 5

0 / 5

0 / 5

0 / 5

0 / 20

Control

0 / 5

0 / 5

0 / 5

0 / 5

0 / 20

0 / 5

0 / 5

0 / 5

0 / 5

0 / 20

Table 6.1.3/2: Measured exposure concentrations and percent of initial concentration of the test item during the definitive test with Daphnia

Sampling date

Fresh medium

0 hours

Old medium

24 hours

Fresh medium

24 hours

Old medium

48 hours

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

Test substance

Calculated concentration1)(µg/L)

Calculated concentration1)(µg/L)

Calculated concentration1)(µg/L)

Calculated concentration1)(µg/L)

Group I

Group II

Group III

Sum2)

Group I

Group II

Group III

Sum2)

Group I

Group II

Group III

Sum2)

Group I

Group II

Group III

Sum2)

10000

0.219

<LOQ

<LOQ

0.219

<LOQ

<LOQ

<LOQ

<LOQ

0.362

0.717

0.304

1.38

0.0624

0.137

<LOQ

0.199

1000

0.608

0.483

0.214

1.31

0.673

0.396

0.163

1.23

0.715

<LOQ

<LOQ

0.715

0.532

<LOQ

<LOQ

0.532

100

0.316

<LOQ

<LOQ

0.316

0.736

0.402

0.188

1.33

0.572

0.372

0.175

1.12

0.518

<LOQ

<LOQ

0.518

Control

<LOQ

<LOQ

<LOQ

<LOQ

0.1023)

<LOQ

<LOQ

-

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

-

1) = 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.

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

3) = 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:
No effects on Daphnia magna were observed in any of the tested WAFs. Therefore, the 48h-EL50 value was determined to be > 10000 mg test item/L, based on nominal loading rates.
Executive summary:

In the acute immobilization test with Daphnia magna, the effects of three water accommodated fractions with nominal loading rates of 100 – 1000 – 10000 mg/L of the test substance were determined at the test facility according to OECD Guideline 202 from 2019 -07 -26 to 2019 -08 -13, with the definitive exposure phase from 2019 -08 -07 to 2019 -08 -09. The test item is a limpid liquid, which is insoluble in water. Since the test item is a UVCB substance with compounds of different water solubility and due to the low aqueous solubility of the test item, the test item solution was prepared as a water accommodated fractions (WAF). This procedure is in accordance with the OECD guidance document No. 23 (2019). Per definition, the exposure has to be expressed in terms of the original loading rate of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF because partly dissolved compounds and mixtures cannot be related to concentrations. The study was performed over a period of 48 hours under semi-static conditions with renewal of test solution after 24 hours as a worst-case exposure scenario. The WAFs were colorless and clear. No undissolved test item was present in any of the tested WAFs. This was checked microscopically (phase-contrast microscope, magnification: 400 times). The test item concentrations were analytically verified via GC-MS/MS at the start of the exposure and at the renewal (0 and 24 hours) and in the old media at the renewal and at the end of the test (24 and 48 hours) in all WAFs and in the control. The ELx-values are based on the nominal loading rates of the test substance, which is recommended by OECD guidance document No. 23 (2019) for testing of WAFs. Since no effects on Daphnia magna were observed in any of the tested WAFs, no calculations were made. The 48h-EL50 value was determined to be > 10000 mg test item/L, based on nominal loading rates.

Description of key information

OECD Guideline 202, GLP, key study, validity 1:

48h-EL50 (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 short-term toxicity of the registered substance to aquatic invertebrates.

In this acute immobilization test with Daphnia magna (NOACK, 2019), the 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 202 with GLP compliance. This study was performed over a period of 48 hours under semi-static conditions with renewal of test solution after 24 hours. No undissolved test item was present in any of the tested WAFs. The test item concentrations were analytically verified via GC-MS/MS at the start of the exposure and at the renewal (0 and 24 hours) and in the old media at the renewal and at the end of the test (24 and 48 hours) in all WAFs and in the control. No effects on Daphnia magna were observed in any of the tested WAFs. Therefore, the 48h-EL50 value was determined to be > 10000 mg test item/L, based on nominal loading rates.

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