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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:
7 July 2009 and 4 September 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern GLP study conducted in accordance with OECD test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
equivalent or similar to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes
Vehicle:
no
Details on test solutions:
Due to the low aqueous solubility and complex nature of the test material for the purposes of the definitive test the test material was prepared as a Water Accommodated Fraction (WAF). An amount of test material (400 mg) was added to the surface of 4 litres of reconstituted
water to give the 100 mg/l loading rate. After the addition of the test material, the reconstituted 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 mixture 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 approximate 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test material to be present.
In the definitive test 250 ml glass jars containing approximately 250 ml of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 22C with a photoperiod of 16 hours light and 8 hours
darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated. The control group was maintained under identical conditions but not exposed to the test material.

Test organisms (species):
Daphnia magna
Details on test organisms:
Juvenile Daphnia Magna ( less than 24 hours old) were taken from healthy laboratory cultures, held and maintain at Harlan Laboratories Ltd. Details of culture maintanence are described in Annex 5 (attached).
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
24 and 48 hours
Hardness:
approximately 250mg/l as CaC03
Test temperature:
22C
pH:
7.9-8.0
Dissolved oxygen:
8.2 TO 8.6 mg O2/l
Salinity:
no data
Nominal and measured concentrations:
100 mg/l loading rate WAF
Details on test conditions:
See Appendix 5, 3. Test Materials and Methods (attached) for details on test condition.

TEST SYSTEM
In the definitive test 250 ml glass jars containing approximately 250 ml of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 22C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated. The control group was maintained under identical conditions but not exposed to the test material.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and the dissolved oxygen concentration were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter and the temperature was measured using a Hanna Instruments HI93510 digital thermometer.

TEST CONCENTRATIONS
- Range finding study- 10 daphnids were exposed to a series of nominal loading rates of 10 and 100 mg/l. The test material was prepared as a WAF.Daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 22C with a photoperiod of 16 hours light
and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. The temperatures were measured to be slightly in excess of the 20 ± 1°C given in the protocol. The control group was maintained under identical conditions but not exposed to the test material.
Based on the results of the range-finding test a Limit test was conducted at a single loading rate of 100 mg/l to confirm that no immobilisation or adverse reactions to exposure were observed.
Reference substance (positive control):
yes
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Validation of Mixing Period- Pre-study investigational work conducted for the validation of mixing period indicated that levels of test material were below the limit of quantitation of the analytical method employed, for a 100 mg/l loading rate WAF, after a stirring period of 23 hours with a 1-Hour standing period. Quantifiable amounts of test material were not detected when the mixing period was extended up to 95 hours with a 1-Hour standing
period. Therefore it was considered appropriate to use a 24 hour preparation period.

Range-finding Test - No significant immobilisation was observed at the 10 and 100 mg/l loading rate WAFs. A single immobilised daphnid was observed at the 100 mg/l loading rate WAF test concentration after 24 hours exposure. This was considered to be due to natural causes rather than a toxic effect given that no further immobilisation was observed and the immobilisation was only 10%. Based on this information, a single loading rate of four replicates, of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test.

Definitive Test- There was no significant immobilisation in 20 daphnids exposed to a 100 mg/l loading rate WAF for a period of 48 hours. The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/l loading rate WAF. The No Observed Effect Loading rate is based upon no significant immobilisation at this loading rate. A single immobilised daphnid was observed at the 100 mg/l loading rate WAF test
concentration after 24 hours exposure. This was considered to be due to natural causes rather than a toxic effect given that no further immobilisation was observed and the immobilisation was less than 10%. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.




Results with reference substance (positive control):
The 24-Hour EC50 for the reference material to Daphnia magna based on nominal concentrations was 1.0 mg/l with 95% confidence limits of 0.90– 1.2mg/l. The 48-Hour EC50 for the reference material to Daphnia magna based on nominal concentrations was 0.78 mg/l with 95% confidence limits of 0.68 – 0.88 mg/l. The No Observed Effect Concentrations after 24 and 48 hours were 0.56 and 0.32 mg/l respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*50 value of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading rate was 100 mg/l loading rate WAF.
Executive summary:

This study was performed to assess the acute toxicity of the test material to Daphnia magna. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test material,

at a single nominal loading rate of 100 mg/l for 48 hours at a temperature of 22C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours. The 48-Hour EL*50 for the test material to Daphnia magna based on nominal loading rates was greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/l loading rate WAF.

Description of key information

A key GLP study reporting 48h EL 50 > 100 mg/l loading rate WAF, OECD, 202.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

In a key GLP compliant study conducted in accordance with the OECD 202 guideline reported that the 48-Hour EL50 for the Daphnia Magna exposed to the test substance (WAF preparations) at the limit of water solubility, was greater than 100 mg/l in terms of the initial test substance loading rate. Correspondingly the No Observed effect Loading rate was 100 mg/l loading rate WAF.