Hatcol 1760

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 12 May 2008 and 19 June 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). Date of inspection: 21/08/2007. Date of signature: 15/10/2007

Test material

Test material information

Constituent 1

Details on test material:

Sponsor's identification :Hatcol 1760
Description : clear colourless slightly viscous liquid
Date received : 4 February 2008
Storage conditions :room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Range finding (nominal loading rate prepared was WAF): 10 and 100 mg/l
definitive test: 100 mg/l (loading rate)

- Sampling method: Water samples were taken from the control (replicates R1 – R4 pooled) and the 100 mg/l loading rate WAF test group (replicates R1 – R2 and R3 – R4 pooled) at 0 and 48 hours for quantitative analysis. The test material concentration in the test samples was determined by high performance liquid chromatography (HPLC) using an external standard. The test material gave a chromatographic profile consisting of multiple peaks. The results have been calculated using the total peak area associated with the test material.
The method was developed by the Department of Analytical Services, Safepharm Laboratories Limited.

- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. 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 Water Accommodated Fraction (WAF) of the test material in cases where the test material is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test material with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test material and water phase. At the completion of mixing, the test material phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test material and/or leachates from the test material). Exposures are expressed in terms of the original concentration of test material in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test material in the WAF.

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. This was stirred for 23 hours. 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 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.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 48 hours (see Appendix 2).


- Eluate:
Stock solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l

- Differential loading: no data

- Controls:
A (negative) control group was maintained under identical conditions but not exposed to the test material.

A positive control conducted approximately every six months used potassium dichromate as the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
An amount of reference material (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/l stock solution. An aliquot (50 ml) of this stock solution was diluted in reconstituted water and the volume adjusted to 500 ml to give a 10 mg/l stock solution. Aliquots (16, 28, 50, 90 and 160 ml) of the 10 mg/l stock solution were each separately dispersed in a final volume of 500 ml of reconstituted water to give the test series of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Exposure conditions for the positive control were similar to those used in the definitive test.
The temperature was maintained at approximately 21°C.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable

- Concentration of vehicle in test medium (stock solution and final test solution): not applicable

- Evidence of undissolved material (e.g. precipitate, surface film, etc): Observations on the test media were carried out during the mixing and testing of the WAFs.
At both the start and end of the mixing period and following the 1-Hour settlement period the WAF was observed to have formed a clear colourless water column with oily globules of test material floating at the water surface. After siphoning and for the duration of the test, the 100 mg/l loading rate was observed to be a clear, colourless solution. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test material to be present.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain: not applicable
- Source: in-house laboratory cultures
- Age at study initiation (mean and range, SD): less than 24 hours old
- Weight at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): no data
- Valve height at study initiation, for shell deposition study (mean and range, SD): not applicable
- Peripheral shell growth removed prior to test initiation: not applicable
- Method of breeding: Parthenogenesis
- Feeding during test: not fed during exposure period


ACCLIMATION
- not applicable

QUARANTINE (wild caught)
- not applicable

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

h

Post exposure observation period:

None

Test conditions

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

Temperature was maintained at 21.1°C to 21.6ºC throughout the test.
Measured using a Hanna Instruments HI 93510 digital thermometer.

pH:

7.9 - 8
Measured using a WTW pH/Oxi 340I pH

Dissolved oxygen:

8.5 mg O2/l at 0 hours
8.3 mg O2/l at 48 hours
Measued using a dissolved oxygen meter.

Salinity:

No data

Nominal and measured concentrations:

In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 10 and 100 mg/l. The test material was prepared as a WAF.

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.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass jars
- Type: closed
- Material, size, headspace, fill volume: 250 ml glass jars containing approximately 250 ml of test preparation
- Aeration: test vessels were not aerated
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): The test preparations were not renewed during the exposure period
- No. of organisms per vessel: 5 daphnida
- No. of vessels per concentration (replicates): 2 (duplicates)
- No. of vessels per control (replicates): 2 (duplicates)
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: not stated in report


TEST MEDIUM / WATER PARAMETERS
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l

An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.

OTHER TEST CONDITIONS
- Adjustment of pH: not conducted during the study
- Photoperiod: The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Light intensity: not stated in report

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
An estimate of the EC50 value at 3 hours was given by inspection of the immobilisation data.
The EC50 value and associated confidence limits at 24 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) and the EC50 value and associated confidence limits at 48 hours and the slope of the response curve and its standard error were calculated by the maximum-likelihood probit nethod (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).
Probit analysis is used where two or more partial responses to exposure are shown.
When only one partial response is shown the trimmed Spearman-Karber method is appropriate.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable, as definitive test is a limit test

- Justification for using less concentrations than requested by guideline: no immobilisation or adverse reactions to exposure during range-finding study.

- Range finding study
In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 10 and 100 mg/l. The test material was prepared as a WAF.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal loading rate

Basis for effect:

other: Immobilisation

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal loading rate

Basis for effect:

other: Immobilisation

Details on results:

- Behavioural abnormalities: Please see data in the following section "Remarks on Results including tables and figures"

- Observations on body length and weight: none. Immobilisation only observed.

- Other biological observations: none

- Mortality of control: immobilisation only observed. Mortality not assessed.

- Other adverse effects control: none stated in report

- Abnormal responses: none stated in report

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Pre-study work (see Appendix 2) indicated that there was a significant increase in the measured test concentrations by extending the preparation period from 24 to 96 hours. However, observations made on the WAF stirred for a period of 95 hours indicated that gross mixing of the test material had occurred. Whilst the aqueous phase was removed and filtered through a glass wool plug it was considered that some undissolved test material may have remained which may have resulted in excessively high measured test concentrations. As such it was considered that a preparation period of 24 hours was sufficient to ensure equilibration between the test material and water phase

- Effect concentrations exceeding solubility of substance in test medium: Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be less than the limit of quantitation (LOQ) of the analytical method employed which was assessed down to 0.41 mg/l.

Results with reference substance (positive control):

- Results with reference substance valid? Yes. Please see data in the following section "Remarks on Results including tables and figures"

- Mortality: not applicable

- EC50/LC50: The No Observed Effect Concentration 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.

The results from the positive control with potassium dichromate were within the normal range for this reference material.

Reported statistics and error estimates:

The EC50 value and associated confidence limits at 24 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) and the EC50 value and associated confidence limits at 48 hours and the slope of the response curve and its standard error were calculated by the maximum-likelihood probit nethod (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).
Probit analysis is used where two or more partial responses to exposure are shown.
When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

Any other information on results incl. tables

there was no immobilisation in 20 daphnids exposed to 100 mg/l loading rate WAF for a period of 48 hours. Inspection of the immobilisation data gave the following results: EL50 at 24 and 48 hours of > 100 mg/l.

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 zero immobilisation at this loading rate.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

*EL = Effective Loading Rate

Cumulative immobilisation data from the exposure of Daphnia magna to the reference material during the positive control are given below. 

Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the trimmed Spearman-Karber method (Hamiltonet al1977) at 24 hours and the probit method (Finney 1971) at 48 hours based on the nominal test concentrations gave the following results:

Time (h)	EC50(mg/l)	95% Confidence limits (mg/l)
3	> 3.2*		-
24	0.97	0.85	-	1.1
48	0.70	0.62	-	0.80

The No Observed Effect Concentration 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.

The slope and standard error of the response curve at 48 hours was 11 (SE = 2.3). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of the response curve at 24 hours.

The results from the positive control with potassium dichromate were within the normal range for this reference material.

*A 3-Hour EC₅₀value was not calculated as less than 50% immobilisation occurred at this time point.

Table1               Cumulative Immobilisation Data in the Range-findingTest

Nominal Loading Rate (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
10	0	0
100	0	0

Table 2               Cumulative Immobilisation Data in the DefinitiveTest

Nominal Loading Rate (mg/l)		Cumulative Immobilised 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              Cumulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	3 Hours				24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0	0	0	0	0
0.56	0	0	0	0	0	0	0	0	1	2	3	15
1.0	0	0	0	0	6	5	11	55	9	10	19	95
1.8	0	0	0	0	10	10	20	100	10	10	20	100
3.2	2	1	3	15	10	10	20	100	10	10	20	100

 

R₁– R₄= Replicates 1 to 4

*= Start of mixing period

+= End of mixing period

R₁– R₂= Replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

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.

Executive summary:

Introduction. A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Directive 92/69/(which constitutes Annex V of Council Directive 67/548/).

Methods. 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 21.1°C to 21.6ºC under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of approximately 21°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results. The 48-Hour EL*₅₀ for the test material toDaphnia 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.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be less than the limit of quantitation (LOQ) of the analytical method employed which was assessed down to 0.41 mg/l.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole, and the dissolved test material was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

The 48-Hour EC₅₀for the reference material to Daphnia magna based on nominal concentrations was 0.70 mg/l with 95% confidence limits of 0.62 – 0.80 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

*EL = Effective Loading Rate