Sodium naphthalene-2-carboxylate

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:

7 August 2008 and 3 October 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Test material

Test material information

Constituent 1

Reference substance name:

Sodium naphthalene-2-carboxylate

EC Number:

700-038-4

Cas Number:

17273-79-9

Molecular formula:

C11 H7 Na O2

IUPAC Name:

Sodium naphthalene-2-carboxylate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l.
- Sampling method: Water samples were taken from the control (replicates R1 – R4 pooled) and the 100 mg/l 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 a single peak.
- Sample storage conditions before analysis:Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
- Sample storage conditions before analysis:

Sponsor's identification : NA-Na
Description : white crystalline solid
Batch number : 7001
Date received : 12 May 2008
Storage conditions : room temperature in the dark

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: . The test material was dissolved directly in water.
- Eluate:

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

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.

- Differential loading: Not applicable
- Controls: The control group was maintained under identical conditions but not exposed to the test material.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): The test material was dissolved directly in water.
- Concentration of vehicle in test medium (stock solution and final test solution): An amount of test material (50 mg) was dissolved in reconstituted water and the volume adjusted to 500 ml to give a 100 mg/l test concentration from which a series of dilutions was made to give further test concentrations of 10, 1.0 and 0.10 mg/l. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: 1st instar
- 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): No Data
- Peripheral shell growth removed prior to test initiation: No Data
- Method of breeding: parthenogenesis
- Feeding during test
- Food type: Each culture was fed daily with a suspension of algae (Chlorella sp.).
- Amount: Not available
- Frequency: Daily


ACCLIMATION
- Acclimation period:Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 21°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 suspension of algae (Chlorella sp.). 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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

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

Test temperature:

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 21°C to 22°C. Temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

pH:

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 pH and dissolved oxygen concentration were measured using a WTW pH/Oxi 340I pH and the dissolved oxygen meter.

Dissolved oxygen:

Dissolved oxygen concentrations were recorded at the start and termination of the test.
Results 8.6mg O2/l.The pH and dissolved oxygen concentration were measured using a WTW pH/Oxi 340I pH and the dissolved oxygen meter.

Salinity:

No data

Nominal and measured concentrations:

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 99% to 101% of the nominal value and so the results are based on nominal test concentrations only.

Nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel:Glass jars
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 ml glass jars filled with 100ml 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:10 daphnids
- No. of vessels per concentration (replicates):5
- No. of vessels per control (replicates):5
- No. of vessels per vehicle control (replicates):5
- Biomass loading rate: Not available


TEST MEDIUM / WATER PARAMETERS
-source/preparation of dilution water: Reconstituted water
- 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

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: None during the test
- Photoperiod:16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods.
- Light intensity:Not stated


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : An estimate of the EC50 values was given at 3 hours by inspection of the immobilisation data.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: 10
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study
- Test concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l.
- Results used to determine the conditions for the definitive study:Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg/l to confirm that at the maximum concentration given in the OECD/EEC Test Guidelines no immobilisation or adverse reactions to exposure were observed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

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:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: Immobilization
- Observations on body length and weight:Not available
- Other biological observations:None observed
- Mortality of control:No data
- Other adverse effects control: No data
- Abnormal responses:No data
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No data
- Effect concentrations exceeding solubility of substance in test medium: Throughout the duration of the test the test concentrations were observed to be clear colourless solutions.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Mortality: Not stated
- EC50/LC50: >100mg/l
- Other:

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

A single immobilised daphnid was observed to be present in two of the 100 mg/l test replicates at 48 hours. This immobilisation was considered to be due to natural causes as no immobilisation was observed in the remaining two test replicates and this immobilisation amounted to 10% which is within the limits allowed for in the Test Guidelines. Inspection of the immobilisation data gave the following results:

Time (h)	EC50(mg/l)	95% Confidence limits (mg/l)
24	> 100	-
48	> 100	-

The No Observed Effect Concentration after 24 and 48 hours exposure was 100 mg/l. The No Observed Effect Concentration is based upon no significant immobilisation at this concentration.

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.

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

Positive Control

Cumulative immobilisation data from the exposure ofDaphnia magnato the reference material (Safepharm Laboratories Project No: 0039/0977) 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 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. The mean 48-Hour EC₅₀value calculated from all positive controls was 0.78 mg/l (sd = 0.22).

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

Table 1-Cumulative Immobilisation Data in the Range-finding Test

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
0.10	0	0
1.0	0	0
10	0	0
100	0	0

 

Table 2-Cumulative Immobilisation Data in the Definitive Test

Nominal Concentration (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
	R2	0			0
	R3	0	0	0	0	0	0
	R4	0			0
100	R1	0	0	0	0	2	10
	R2	0			0
	R3	0			1[1]
	R4	0			1*

R₁– R₄= Replicates 1 to 4

Table 3-Cumulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (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– R₂= Replicates 1 and 2

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 EC50 of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.

Executive summary:

Introduction.A study was performed to assess the acute toxicity of the test material toDaphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphniasp, 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 an aqueous solution of the test material at a concentration of 100 mg/l for 48 hours at a temperature of 21°C to 22°C 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 magnawas 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 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 EC₅₀for the test material toDaphnia magnabased on nominal test concentrations was greater than 100 mg/l and correspondingly the No Observed Effect Concentration was 100 mg/l.

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

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 99% to 101% of the nominal value and so the results are based on nominal test concentrations only.

The 48-Hour EC₅₀for the reference material toDaphnia magnabased 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.