Ammonium sulphamidate

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 1 June 2011 and 15 July 2011.

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.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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)

Version / remarks:

of Commission Regulation (EC) 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium sulphamate

IUPAC Name:

Sodium sulphamate

Details on test material:

Sponsor's identification: Sodium sulphamate
CAS No : 13845-18-6
Identifier : TIS I0442
Description : white solid
Purity : 99.67%
Batch number : LE12568
Date received : 6 May 2011
Expiry date : 6 May 2013
Storage conditions: room temperature in the dark over silica gel

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Single concentration 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 method of analysis, recovery and test preparation analyses are described in Appendix 5 Verification of Test Concentrations (see attached background material).

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

Test solutions

Vehicle:

no

Details on test solutions:

Range-finding test:
The test concentration 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 test concentrations of 0.010, 0.10, 1.0, 10 and 100 mg/l. The test item was dissolved directly in reconstituted water.

An amount of test item (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give the 100 mg/l test concentration from which serial dilutions were prepared, in reconstituted water, to give the remainder of the test series of 10, 1.0, 0.10 and 0.010 mg/l.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 20°C to 22°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. Some of the temperatures were measured to be slightly in excess of the 20 ± 1°C given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed throughout the duration of the test and that the temperatures were within the test guideline specification. 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 immobilised Daphnia magna were recorded.

A sample of each test concentration 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 at approximately -20°C prior to analysis. Only samples covering the concentrations to be used in the definitive test were analysed.

Definitive test:
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/EC Test Guidelines, no immobilisation or adverse reactions to exposure were observed.

Experimental preparation

For the purpose of the definitive test the test item was dissolved directly in reconstituted water.
An amount of test item (200 mg) was dissolved in reconstituted water and the volume adjusted to 2 litres to give the 100 mg/l test concentration.
The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours.

-Controls: The control group was maintained under identical conditions but not exposed to the test item.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test Species:
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 (seedetails below) in a temperature controlled room at 21°C to 22°C. A temperature at which the stock daphnids were kept was observed to be slightly in excess of the range given in the study plan of 20 ± 1°C. This deviation was considered not to have affected the outcome or the validity of the test as there was no effect on survival and reproduction of the stock cultures and the temperature range was within guideline specification. 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 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.

Reconstituted Water – Elendt M7 Medium
Solution Concentration of stock solution (mg/l)
(I) H3BO3 57190
(II) MnCl2.4H2O 7210
(III) LiCl 6120
(IV) RbCl 1420
(V) SrCl2.6H2O 3040
(VI) NaBr 320
(VII) Na2MoO4.2H2O 1260
(VIII) CuCl2.2H2O 335
(IX) ZnCl2 260
(X) CoCl2.6H2O 200
(XI) Kl 65
(XII) Na2SeO3 43.8
(XIII) NH4VO3 11.5
(XIV) Na2EDTA.2H2O 5000
FeSO4.7H2O 1991
An aliquot (dependant on the volume of medium required) of each stock solution was added to a final volume of deionised reverse osmosis water to give stock solution A and stored at approximately 21ºC.

Macro Nutrient Stock Solutions

Solution Concentration of stock solution (g/l)
(I) CaCl2.2H2O 293.80
(II) NaHCO3 64.80
(III) MgSO4.7H2O 246.60
(IV) Na2SiO3.9H2O 50.00
(V) KCl 58.00
(VI) NaNO3 2.74
(VII) K2HPO4 1.84
(VIII) KH2PO4 1.43

Vitamin Nutrients

Solution Concentration of stock solution (mg/l)
(IX) Thiamine hydrochloride 750
Cyanocobalamine (vitamin
B12) 10.0
D(+) biotin (vitamin H) 7.5
The final medium was prepared by adding an aliquot of stock solution A along with aliquots of each individual Macro Nutrient Stock Solution and an aliquot of the vitamin nutrient to the required amount (final volume) of deionised reverse osmosis water.
The pH of the prepared media was 8.0 ± 0.2 and stored at approximately 21ºC.

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:

Water temperature was recorded daily throughout the test and was measured using a Hanna Instruments HI 93510 digital thermometer.
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 study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed throughout the duration of the test and that the temperatures were within the test guideline specification.

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 a Hach HQ30d Flexi Handheld meter
There were no treatment related differences for pH.

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 Hach HQ30d Flexi Handheld meter.
The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.

Salinity:

Freshwater used.

Nominal and measured concentrations:

In the range-finding test Daphnia magna were exposed to a series of nominal concentrations of 0.010, 0.10, 1.0, 10 and 100 mg/l.
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg/l.

Details on test conditions:

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 21°C to 22°C 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 item.

The test preparations were not renewed during the exposure period.

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
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg/l for the definitive test

TEST WATER
Reconstituted water used for both the range-finding and definitive test is defined in below.


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.

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:

other: immobilisation

Remarks on result:

other: 95% CL -no stated

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:

other: immobilisation

Remarks on result:

other: 95% CL not stated

Details on results:

Range-finding Test :
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1 (any other information on results incl. tables section section).

No immobilisation was observed at the test concentrations employed in the test.

Based on this information, a single test concentration of four replicates, of 100 mg/l was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EC Test Guidelines, no immobilisation or adverse reactions to exposure were observed.

Chemical analysis of the 100 mg/l test preparation at 0 and 48 hours (see attached background material) showed measured concentrations to be 101% and 98% respectively indicating that the test item was stable under test conditions.


Definitive Test:
Immobilisation data
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2 (any other information on results incl. tables section section).

There was no immobilisation in 20 daphnids exposed to a test concentration of 100 mg/l for a period of 48 hours. Inspection of the immobilisation data gave the following results:
Time (h) EC50 (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 zero immobilisation at this concentration.

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

Observations on test item solubility:
The test preparations were observed to be clear, colourless solutions throughout the duration of the test.

Physico-chemical measurements:
The results of the physico-chemical measurements are given below. Temperature was maintained at 21°C to 22°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Some of the temperatures were measured to be slightly in excess of the 20 ± 1°C given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed throughout the duration of the test and that the temperatures were within the test guideline specification.
The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.


Verification of test concentrations:
Analysis of the test preparations at 0 and 48 hours (see attached background material) showed measured test concentrations to range from 90% to 105% of nominal value and so it was considered justifiable to estimate the EC50 values in terms of the nominal test concentrations only.

Validation Criteria:

In the control, no daphnia became immobilised. Thus the validity criterion was met.
The oxygen content was ≥ 8.7 mg/l in the control and test vessels at the end of the test. Thus the validity criterion was met.

Results with reference substance (positive control):

Positive Control

A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
Exposure conditions for the positive control were similar to those in the definitive test.
Analysis of the immobilisation data by the maximum-likelihood probit method (Finney 1971 ) at 24 and 48 hours based on the nominal test concentrations gave the following results:

24 hr EC50: 1.5 mg/l (95% Confidence limits 1.3 - 1.8 mg/l)
48 hr EC50: 0.99 mg/l (95% Confidence limits 0.85 - 1.1 mg/l)

The No Observed Effect Concentration after 24 and 48 hours was 0.56 mg/l. 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 item.

Reported statistics and error estimates:

None perfromed

Any other information on results incl. tables

Table 1              Cumulative Immobilisation Data in the Range-finding Test

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
0.010	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	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

 

R₁– R₄= Replicates 1 to 4

Physico-Chemical Measurements

Nominal Concentration (mg/l)		0 Hours				24 Hours	48 Hours
		pH	mg O2/l	%*	T°C	TºC	pH	mg O2/l	%*	T°C
Control	R1	7.8	9.2	103	21	22	7.8	8.8	101	22
	R2	7.7	9.2	103	21	22	7.8	8.8	101	22
	R3	7.7	9.2	103	21	22	7.7	8.7	100	22
	R4	7.7	9.2	103	21	22	7.7	8.7	100	22
100	R1	7.7	9.1	102	21	22	7.7	8.7	100	22
	R2	7.6	9.2	103	21	22	7.7	8.7	100	22
	R3	7.7	9.1	102	21	22	7.7	8.7	100	22
	R4	7.7	9.1	102	21	22	7.8	8.7	100	22

*ASV= Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

R₁– R₄= Replicates 1 to 4

Applicant's summary and conclusion

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

Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item 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.

Results.

The 48-Hour EC₅₀ for the test item to Daphnia magna based on nominal test concentrations was greater than 100 mg/l. 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 90% to 105% of nominal value and so the results are based on nominal test concentrations only.