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Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-11-20 - 2018-03-09 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals (April 2004) No 202 "Daphnia sp., Acute Immobilisation Test"

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Method C.2 of Commission Regulation (EC) No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature (15 to 25 ºC) without protection from light

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each loading rate WAF test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the old or expired pooled replicates (R1 to R4) at 24 and 48 hours for quantitative analysis. Samples were stored frozen prior to analysis.
Duplicate samples were taken and stored frozen for further analysis if necessary.
Only samples at the NOEL rate and above were analyzed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a WAF of the test item.
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 0.43, 0.94, 2.1, 4.5 and 10 mg/L.
Nominal amounts of test item (9.5, 20.7, 10.5, 22.5 and 50 mg) were each separately added to the surface of 22, 22, 5, 5 and 5 liters of test water respectively to give the 0.43, 0.94, 2.1, 4.5 and 10 mg/L loading rates. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid depth siphoning (the first 75 to 100 mL discarded) to give the 0.43, 0.94, 2.1, 4.5 and 10 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro dispersions or undissolved test item to be present.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
- Feeding during test: no

ACCLIMATION
- Acclimation period: none

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Test temperature:

Test vessels were maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C

pH:

The pH of the prepared media was 7.9 ± 0.3.
Measured: 7.9 - 8.0

Dissolved oxygen:

8.6 - 9.1 mg/l

Nominal and measured concentrations:

Nominal: 0, 0.43, 0.94, 2.1, 4.5, 10 mg/l
Measured: See below, results section & attachment

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass jars containing approximately 100 mL of test preparation were used.
- Type (delete if not applicable): The test vessels were then covered to reduce evaporation
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): after 24h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: between 200 and 1200 lux

Water Quality Criteria
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24 Hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
The light intensity during the light period was measured using an ATP Instrumentation Lux meter.
The appearance of the test media was recorded daily.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

RANGE-FINDING STUDY
- Test concentrations: In the range finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study:
No significant immobilization was observed at the 1.0 mg/L loading rate WAF, however, immobilization was observed at 10 and 100 mg/L loading rate WAF.
Sub lethal effects of exposure were observed in all test concentrations. These responses were reduced movement and trapping at the surface.
Based on this information loading rates of 0.43, 0.94, 2.1, 4.5 and 10 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

A positive control (separate study) used potassium dichromate as the reference item

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

4.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Remarks:

NOEL

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOELR

Remarks:

LOEL

Effect conc.:

4.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: Sub lethal effects of exposure were observed in all test concentrations. These responses were trapping at the surface and reduced movement.
- Mortality of control: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Substance was applied as WAF

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50(48h) = 0.75 mg/l
- Limit test: no
- Dose-response test: yes
- ECx: EC50(48h) = 0.75 mg/l
- Other:
A positive control stzdy used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.
Analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:
Time Point (Hours) EC50 (mg/L) 95% Confidence Limits (mg/L) NOEC (mg/L) LOEC(mg/L)
24 0.79 0.73 - 0.86 0.56 1.0
48 0.75 0.56 - 1.0 0.56 1.0

The NOEC is based upon equal to or less than 10% immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item. The results of the inter laboratory tests and a Technical Corrigendum to ISO 6341 give a 24 Hour EC50 for potassium dichromate within the range 0.6 mg/L to 2.1 mg/L

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD guideline 202 and EU method C.2 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the substance towards daphnids. In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a WAF of the test item. Chemical analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.057 to 6.2 mg/L. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations to range from 0.053 to 5.1 mg/L.
The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.
The EL50(48h) was determined to be 4.9 mg/ml. According to Regulation (EC) No.1272/2008 and amendments (CLP), the criteria for classification of a substance in category Acute 1 are defined on the basis of acute aquatic toxicity data only (EC50, EL50, LC50 or LL50); classification is required for values ≤ 1 mg/L. Therefore, the substance does not need to be classified for acute (short-term) aquatic hazard. However, according to Table 4.1.0 (Commission Regulation (EU) No 286/2011 of 10 March 2011), Classification categories for hazardous to the aquatic environment, (iii) Substances for which adequate chronic toxicity data are not available, and the substance is not rapidly degradable and/or the experimentally determined BCF ≥ 500 (or, if absent, the log K ow ≥ 4), classification as Category Chronic 2 is required if the following short-term results are obtained:
96 hr LC 50 (for fish) > 1 to ≤10 mg/l and/or
48 hr EC 50 (for crustacea) > 1 to ≤10 mg/l and/or
72 or 96 hr ErC 50 (for algae or other aquatic plants) > 1 to ≤10 mg/l
With an EL50(48h) of 4.9 mg/ml, and the fact that no biodegradation observed, 0% (O2 consumption) in 28 days (OECD TG 301D), the substance must be classified as Aquat. Chron. Cat. 2.

Executive summary:

Introduction

A GLP study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

 

Methods

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range‑finding test, 20 daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 0.43, 0.94, 2.1, 4.5 and 10 mg/L for 48 hours at a temperature of 20 to 22 °C under semi-static test conditions. The number of immobilized daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

Results

Chemical analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.057 to 6.2 mg/L. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations to range from 0.053 to 5.1 mg/L.

The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Daphnia magna to the test item gave the following results:

 

Time Point (Hours)	EL50 (mg/L Loading RateWAF)	95% Confidence Limits (mg/L Loading Rate WAF)	NOEL (mg/L)	LOEL (mg/L)
48	4.9	Not determined due to the nature of the data	2.1	4.5

NOEL  = No Observed Effect Loading Rate

LOEL   = Lowest Observed Effect Loading Rate

The substance must be classified as Aquat. Chron. Cat. 2.

Description of key information

Acute Toxicity to Aquatic invertebrates: EL50(48h) = 4.9 mg/l, NOEL(48h) = 2.1 mg/l, LOEL(48h) = 4.5 mg/l for Daphnia magna (WAF, semi-statically, OECD 202, EU method C.2, GLP)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

4.9 mg/L