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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:
17 September 2013 to 07 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in accordance with current OECD guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
See "Principles of method if other than guideline" for details of deviations.
Principles of method if other than guideline:
Deviation No.1 (24 October 2013)In the range finding test at 0 hours the recorded temperature in the control vessel was higher than the temperature recommended in the OECD test guideline. The recorded temperature was 23 °C and the recommended maximum is 22 °C, however as there was no immobilization or sub-lethal effects noted this is considered not to have adversely affected the results of the test.Deviation No.2 (24 October 2013)In the definitive test at 48 hours the light intensity was not measured in error. This is considered not to have adversely affected the results of the test.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):Not applicable
Analytical monitoring:
yes
Details on sampling:
A sample of each loading rate WAF was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.
Vehicle:
no
Details on test solutions:
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 Water Accommodated Fraction (WAF) of the test item.
Amounts of test item (20, 20 and 200 mg) were each separately added to the surface of 20, 2.0 and 2.0 liters of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. 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. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (seeAppendix 1) in a temperature controlled room at approximately 20 °C. The lighting cycle wascontrolled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusktransition 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.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
Approximate theoretical total hardness of 250 mg/L as CaCO3.
Test temperature:
20 to 22 °C
pH:
pH of 7.8 ± 0.2
Dissolved oxygen:
8.2 to 8.7 mg O2/L
Salinity:
Not applicable
Nominal and measured concentrations:
Range finding test: 1.0, 10, 100 mg/l [nominal loading rate]Main test: 100 mg/l [nominal loading rate]
Details on test conditions:
Procedure
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 Water Accommodated Fraction (WAF) of the test item.
Validation of Mixing Period
Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.
Range-finding Test
The loading rate to be used in the definitive test was determined by a preliminary range-finding test.In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L.Amounts of test item (20, 20 and 200 mg) were each separately added to the surface of 20, 2.0 and 2.0 liters of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. 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. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs.In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 20 to 23 °C 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. Each 250 mL test and control vessel contained 200 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized Daphnia magna were recorded.The control group was maintained under identical conditions but not exposed to the test item.A sample of each loading rate WAF was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.Definitive TestBased 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 immobilization or adverse reactions to exposure were observed.

Experimental Preparation
An amount of test item (250 mg) was added to the surface of 2.5 liters of test water to give the 100 mg/L loading rate. 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 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. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. 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.The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours.

Exposure Conditions
As in the range-finding test 250 mL glass jars containing approximately 200 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 20 to 22 °C with a photoperiod of 16 hours light (661 to 878 lux) 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 item. The test preparations were not renewed during the exposure period.

Evaluations
Test Organism Observations
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 for approximately 15 seconds after gentle agitation.

Water Quality Criteria
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 dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period.

Chemical Analysis of Test Loading Rates
Duplicate water samples were taken from the control and the 100 mg/L loading rate WAF test group (replicates R1 – R4 pooled) at 0 and 48 hours for quantitative analysis. Samples were stored frozen prior to analysis. Duplicate samples were retained frozen for further analysis if necessary

Statistical Analysis
An estimate of the EL50 values was given by inspection of the immobilization data.Validation CriteriaThe results of the test are considered valid if the following performance criteria are met:• No more than 10% of the control daphnids show immobilization or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water).• The dissolved oxygen concentration at the end of the test is >= 3 mg/L in the control and test vessels.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF.
Basis for effect:
mortality
Details on results:
Validation of Mixing Period
Pre-study investigational work indicated that there was no significant increase in the amount of dissolved test item when the preparation period was extended for longer than 24 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

Range-finding Test
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1.No immobilization was observed at 1.0 , 10 and 100 mg/L loading rate WAF.Based on this information, a single loading rate of four replicates, of 100 mg/L, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilization or adverse reactions to exposure were observed.Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.Definitive Test

Chemical Analysis of Test Loading Rates
Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of 0.0438 mg/L and less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.0349 mg/L.The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Immobilization Data
There was no immobilization in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours. Inspection of the immobilization data gave EL50 (mg/L Loading Rate WAF) Gave 24 and 48 values of > 100 mg/l. The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/L loading rate WAF.It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Validation Criteria
The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.

Water Quality Criteria
The results of the water quality measurements are given in Table 3. Temperature was maintained at 20 to 22 ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion.

Observations on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the WAF.At the start of the mixing period the 100 mg/L loading rate was observed to be a clear colorless water column with oily globules of test item floating on the water surface. After 23 hours stirring and a 1-Hour standing period the 100 mg/L loading rate was observed to remain a clear colorless water column with oily globules of test item floating on the water surface. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the 100 mg/L loading rate was observed to be a clear, colorless solution.
Results with reference substance (positive control):
The results from the positive control with potassium dichromate were within the normal range for this reference item.
Reported statistics and error estimates:
None reported

Table 1     Cumulative Immobilization Data in the Range-finding Test

Nominal Loading Rate
(mg/L)

Cumulative Immobilized Daphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Control

0

0

1.0

0

0

10

0

0

100

0

0

 

Table 2     Cumulative Immobilization Data in the Definitive Test

Nominal Loading Rate
(mg/L)

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

24 Hours

48 Hours

No. Per

Replicate

Total

%

No. Per

Replicate

Total

%

Control

R1

0

0

0

0

0

0

R2

0

0

R3

0

0

R4

0

0

100

R1

0

0

0

0

0

0

R2

0

0

R3

0

0

R4

0

0

 

Table 3          Water Quality Measurements

Nominal
Loading Rate
(mg/L)

0 Hours

24 Hours

48 Hours

pH

mg O2/L

T°C

T ºC

pH

mg O2/L

T°C

Control

R1

7.8

8.5

21

21

7.8

8.6

21

R2

7.8

8.3

21

21

7.9

8.7

20

R3

7.8

8.2

22

21

8.0

8.7

21

R4

7.8

8.2

22

21

8.0

8.7

21

100

R1

7.8

8.5

22

21

8.0

8.7

21

R2

7.8

8.5

22

21

8.0

8.7

21

R3

7.8

8.5

22

21

8.0

8.7

21

R4

7.9

8.6

22

21

8.0

8.7

21


R1– R4= Replicates 1 to 4

R1– R4= Replicates 1 to 4

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.
Executive summary:

A Klimish one rated GLP study conducted in accordance with current OECD guidelines is available to assess the effects of acute toxicity to Daphnia Magna. The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.

No classification is applicable.

Description of key information

Assessment of acute toxicity to Daphnia

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

A Klimish one rated GLP study conducted in accordance with current OECD guidelines is available to assess the effects of acute toxicity to Daphnia Magna. The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF. This category of substances does not demonstrate any significant toxicity to aquatic organisms, nor does it contain significant impurities that could effect the outcome of the a specific study.

No classification is applicable.