Eldew APS-307

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 10 February 2009 and 13 March 2009. The positive control was conducted between 9 December 2008 and 11 December 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 the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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:

Date of GLP inspection: 19/08/2008 Date of Signature on GLP certificate: 04/03/2009

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): Eldew APS-307
- Substance type: UVCB (Organic)
- Physical state: pale yellow waxy solid block
- Lot/batch No.: 809114
- Date received: 22 December 2008
- Stability under test conditions: Stable
- Storage condition of test material: room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nominal WAF Loading rate or 100 mg/L
Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis.

- Sampling method:
Water samples were taken from the control (replicates R1 – R2 pooled) and each loading rate WAF test group (replicates R1 - R2 pooled) at 0 and 48 hours (see Appendix 3). Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

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

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Range-finding test:
Due to the low aqueous solubility and complex nature of the test material for thepurposes of the definitive test the test material was prepared as a Water Accommodated Fraction (WAF).The test material was prepared as a Water Accommodated Fraction (WAF). Amounts of test material (2.5, 25 and 250 mg) were each separately added to the surface of 2.5 litres of reconstituted water to give the 1.0, 10 and 100 mg/l loading rates respectively. 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 slight dimple at the water surface. Thestirring 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 and the WAF removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 1.0, 10 and 100 mg/l loading rate WAFs. Microscopic observations of the WAFs were performed and showed there to be no micro-dispersions of test material present.

Definitive Test:
For the purpose of the definitive test the test material was prepared using a Water Accommodated Fraction (WAF). An amount of test material (450 mg ) was added to the surface of 4.5 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 slight 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 tub and pushed through the Nescofilm seal and the WAF removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic observations of the WAF were performed and showed there to be no micro-dispersions of test material 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 3).


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.

- Eluate:
Not applicable

- Controls:
A positive control (Harlan Laboratories Ltd Project No. 0039/1069) 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 20°C.

- Chemical name of vehicle : Reconstituted water

- Concentration of vehicle in test medium:
Not applicable

- Evidence of undissolved material :
Microscopic inspection of the WAFs 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

- Source:
Derived from in-house laboratory cultures.

- Age at study initiation:
Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.

- Feeding during test:
Received no food during exposure

ACCLIMATION
- Acclimation period:
The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.


- Acclimation conditions:
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20 °C.


- Type and amount of food:
Each culture was fed daily with a suspension of algae (Chlorella sp.).
The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

- Health during acclimation:
No mortality observed


Test Water:
The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

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 °C to 22 °C throughout the test.
Some of the temperatures were measured to be slightly in excess of the 20 ± 1°C given in the protocol. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification.
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

pH:

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
The pH was measured using a WTW pH/Oxi 340I pH meter.
There were no treatment related differences for pH.
See Appendix 4 for results.

Dissolved oxygen:

The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration was measured using a dissolved oxygen meter.
See Appendix 4 for results.

Salinity:

Not applicable

Nominal and measured concentrations:

In the range-finding test Daphnia magna were exposed to a series of nominal loading rates WAF of 1.0, 10 and 100 mg/l.
Based on the results of the range-finding test the following loading rate was assigned to the definitive test: 100 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel:
In the definitive test 250 ml glass jars containing approximately 250 ml of test preparation were used. At the start of the study 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 21°C in the dark. 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.

The test preparations were not renewed during the exposure period. Any immobilisation 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 immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

- Type (delete if not applicable): closed

- Material, size, headspace, fill volume: The test vessels were then sealed, with minimal headspace to prevent losses of the test material due to its volatile nature,

- Aeration: None.

- Type of flow-through (e.g. peristaltic or proportional diluter): None.

- Renewal rate of test solution (frequency/flow rate): None.

- No. of organisms per vessel: 5 daphnids were placed in each test and control vessel at random, in the test preparations

- No. of vessels per concentration (replicates): 4 test vessels were used for each test and control group.

- No. of vessels per control (replicates): 4 test vessels were used for each test and control group.


- Source/preparation of dilution water:

Appendix 1 Reconstituted Water
i) 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


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


OTHER TEST CONDITIONS

- Adjustment of pH:
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.

- Photoperiod: A photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.

- Light intensity: Not recorded

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilisation 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 immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS

- Spacing factor for test concentrations:
Based on the results of the range-finding test the following loading rate was assigned to the definitive test: 100 mg/l.

- Justification for using less concentrations than requested by guideline: Not applicable

- Range finding study

- Test concentrations: loading rates of 1.0, 10 and 100 mg/l.

- Results used to determine the conditions for the definitive study:

The loading rates to be used in the definitive test were 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.

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:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL not stated

Details on results:

- Behavioural abnormalities: Not recorded
- Other biological observations: None recorded

- Other adverse effects control: No other effects observed.

- Abnormal responses: None recorded

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None recorded

- Effect concentrations exceeding solubility of substance in test medium: 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 standing period WAFs were observed to have formed clear colourless water columns with an oily slick of test material at the water surface. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present.

Throughout the duration of the test all loading rates were observed to be clear, colourless solutions.

Results with reference substance (positive control):

- Results with reference substance valid? Yes

- Mortality: No mortalities recorded.

- Immobilisation of control:
Cumulative immobilisation data from the exposure of Daphnia magna to the reference material (Harlan Laboratories Ltd Project No. 0039/1069) during the positive control are given in table 3. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 1 and 2 (see attachment 2 in attached background material).

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

Time (h) EL50 (mg/l) 95% Confidence limits (mg/l) 3 >3.2 -
24 0.82 0.71 - 0.94
48 0.71 0.61 - 0.81

The No Observed Effect Concentration after 24 and 48 hours was 0.32 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

The slopes and their standard errors of the response curves at 24 and 48 hours were 8.1 (SE = 1.7) and 8.6 (SE = 1.8) respectively.

The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/l (sd = 0.21).

Reported statistics and error estimates:

An estimate of the EC50 value at 3 hours was given by inspection of the immobilisation data.
The EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and standard error of the response curves were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).

Probit analysis is used where two or more partial responses to exposure are shown.

Any other information on results incl. tables

RESULTS

Validation of Mixing Period

Pre-study work (see appendix 2) indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 hrs.

Range-finding Test

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in Table 1. 

No immobilisation was observed throughout the test.

Based on this information, a single test loading rate of four replicates, of 100mg/l was selected for the definitive test.

DefinitiveTest

Immobilisation data

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2. 

There was no significant immobilisation in 20 daphnids exposed to a loading rate of 100 mg/l for a period of 48 hours. Inspection of the immobilisation data gave the following results:

Time (h)	EL*50(mg/l)	95% Confidence limits (mg/l)
24	>100	-
48	>100	-

*EL = Effective Loading Rate

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 daphnia was observed in the controls and two immobilised daphnids were observed in the 100 mg/l loading rate WAF test concentration. These immobilised daphnids were considered to be due to natural causes and not a result of toxicity.

Physico-chemical Measurements

The results of the physico-chemical measurements are given in Appendix 4.

Temperatures in the control and test vessels were maintained at 21°C to 22°C throughout the test. Some of the temperatures were slightly in excess of the 20 ± 1°C given in the protocol. This was considered not to affect the results of the test as no significant adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification. There were no treatment related differences for pH or oxygen concentration observed throughout the test.

Verification of Test Concentrations

Analysis of the test preparations at 0 and 48 hours (see appendix 3) showed measured test concentrations at 0 hours to range from 0.0126 to 0.0148 mg/l and less than the limit of quantitation of the analytical method at 48 hours. Given that 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 very low, the results were based on nominal loading rates only.

Positive Control

Cumulative immobilisation data from the exposure of Daphnia magna to the reference material (Harlan Laboratories Ltd Project No. 0039/1069) during the positive control are given in Table 3. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 1 and 2. Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the probit method (Finney 1971) at 24 and 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	0.82	0.71 - 0.94
48	0.71	0.61 - 0.81

The No Observed Effect Concentration after 24 and 48 hours was 0.32 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration. The slopes and their standard errors of the response curves at 24 and 48 hours were 8.1 (SE = 1.7) and 8.6 (SE = 1.8) respectively.

The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC₅₀ value calculated from all positive controls was 0.78 mg/l (sd = 0.21).

Table 1. Cumulative lmmobilisation Data in the Range-finding Test

Nominal Loading Rate (mg/l)	Cumulative lmmobilised 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 lmmobilisation Data in the Definitive Test

Nominal Loading Rate (mg/l)		Cumulative lmmobilised Daphnia (Initial Population: 5 Per Replicate)
		24 hours			48 hours
		No. Per Replicate	Total	%	No. Per Replicate	Total	%
Control	R1	0	1	5	0	1	5*
	R2	0			0
	R3	0			0
	R4	1			1
100	R1	0	0	0	0	2	10*
	R2	0			1
	R3	0			1
	R4	0			0

R₁ - R₄ = Replicates 1 to 4

* lmmobilisation considered not to be significant as not greater than 10% observed.

Table 3. Cumulative lmmobilisation 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	1	1	2	10	2	2	4	20
1.0	0	0	0	0	7	8	15	75	9	9	18	90
1.8	0	0	0	0	10	10	20	100	10	10	20	100
3.2	0	0	0	0	10	10	20	100	10	10	20	100

R₁ - R₂ = Replicates 1 and 2

Table 4. Vortex Depth Measurements at the Start and End of the Mixing Period

	Nominal Loading Rate (mg/l)
	Control		100
	*	+	*	+
Height of Media Column (cm)	22	22	22	22
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present

* = Start of mixing period

+ = End of mixing period

Applicant's summary and conclusion

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 *EL50 of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading rate was 100 mg/l loading rate WAF.

*EL = Effective Loading rate

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 lmmobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

METHOD:

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a concentration of 100 mg/l for 48 hours at a temperature of 21 to 22°C under static test conditions. lmmobilisation 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 20°C under static test conditions. lmmobilisation and any adverse reactions to exposure were recorded after 3,24 and 48 hours.

RESULT :

The 48-Hour *EL₅₀ 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.

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

Analysis of the test preparations showed measured test concentrations to range from 0.0126 to 0.0148 mg/l at 0 hours and less than the limit of quantitation of the analytical method at 48 hours.

Given that 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 concentration was very low, the results were based on nominal loading rates only.

The 48-Hour EC₅₀ for the reference material to Daphnia magna based on nominal concentrations was 0.71 mg/l with 95% confidence limits of 0.61 - 0.81 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

CONCLUSION:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour *EL₅₀ of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading rate was 100 mg/l loading rate WAF.

*EL₅₀ = Effective loarding rate