1-Naphthol, reaction products with formaldehyde

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 September 2005 and 09 December 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- During preliminary work a compound specific analytical method could not be developed for the test material. Therefore, it was considered appropriate to analyse the test concentrations during the definitive test using Total Organic Carbon (TOC) analysis as higher concentrations of test material could be detected using this method.
- Samples of the control and each test concentration were taken at 0 (fresh media) and 48 hours (old media) for Total Organic Carbon (TOC) analysis.
- Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

EXPERIMENTAL PERPARATION
-Data supplied by the sponsor indicated that the test material contained an active ingredient content of approximately 17 % dissolved in water (approximately 80 %) and sodium hydroxide (approximately 3 %). Although the test material was supplied as a solution in water, preliminary solubility work showed that it was not soluble in reconstituted water used for the test and that a precipitate was formed n addition of the test material to this medium. Therefore, due to the low aqueous solubility of the test material in reconstituted water the test concentrations used in the definitive test were prepared by diluting (with reconstituted water) a saturated solution prepared from initial test material dispersion at a concentration of 100 mg/L. Information supplied by the Sponsor indicated that the solubility of the test material in the diluent could be increased at higher pH values. Therefore, given this information and flowing advice from the competent regulatory authority, the test material was initially prepared in reconstituted water with a pH value of 11. The pH was then re-adjusted to a value to 9 to fall within the tolerance levels of the test organism.
- An amount of the test material (200 mg) was dispersed in 2 L of reconstitute water, with the pH adjusted to 11 using 1 M sodium hydroxide, to give a 100 mg/L stock concentration which was observed to be a clear blue solution. The pH of this was then adjusted to 9 using 1 M hydrochloric acid and any undissolved test material removed by filtration through a 0.2 µm Acrocap filter (first 50 mL discarded to pre-condition the filter) to give a 100 % v/v solution. Aliquots (10, 18, 32, 56, 100, 180, 320 and 560 mL) of the 100 % v/v saturated solution were each separately diluted in a final volume of 1 L of reconstituted water at pH 9 to give the 1.0 ,1.8, 3.2, 5.6, 10, 18, 32 and 56 % v/v saturated solution respectively. Media preparation was conducted under a non-actinic light source and the test vessels were shielded from the light as preliminary chemical analysis indicated that the test material may be unstable in the light.
- Each concentration was inverted several times to ensure adequate mixing and homogeneity.
- During preliminary chemical analysis a compound-specific analytical method could not be developed to detect levels of test material present in a saturated solution of the test material. Therefore the concentration of the test material in the test preparations was developed by Total Organic Carbon (TOC) analysis at 0 and 48 hours.

SOLUBILITY TRIALS
- Initial solubility work indicated that the test material was more soluble in deionised reverse osmosis water than it was in reconstituted water by visual inspection. Therefore, a saturated solution of the test material was prepared to determine the amount of test material that could be dissolved.
- An amount of test material (2500 mg) was added to 2.5 litres of reconstituted water and stirred by magnetic stirrer at approximately 200 rpm at approximately 22 °C for 48 hours. On completion of stirring the mixture was filtered through a 0.2 µm Sartopore filter (initial 1 litre discarded to pre­condition the filter) to give a 100 % v/v saturated solution of the test material. Serial dilutions were prepared from the saturated solution in reconstituted water to give further concentrations of 10 and 1.0 % v/v saturated solution. Samples of the prepared concentrations were taken for recovery and stability analyses. However, the results from analysis showed that concentrations of the test material in the saturated solution were less than the limit of quantitation of the analytical method.
- As the test material could not be detected in the saturated solution a second preparation was conducted in a similar manner at an identical concentration to verify the findings of the initial analysis of a saturated solution of the test material.
- Information provided by the Sponsor indicated that the test material would be more soluble at higher pH values and following further information from the Sponsor it was considered that at lower pH values the test material may change ionic form to a low water soluble protonated form. Therefore, further work including the range-finding test detailed below was conducted using reconstituted water adjusted to pH 9 in order to maximise the amount of dissolved test material for exposure.

PREPARATION AND APPLICATION OF TEST SOLUTION
- Range-finding study: An amount of test material (100 mg) was dispersed in 1 L of reconstituted water, with a pH of approximately 8 to give a 100 mg/L stock dispersion. Any dissolved test material was removed by filtration through a 0.2 µm Acrocap filter (first approximately 50 mL discarded to pre-condition the filter) to prepare the 100 % v/v saturated solution.
A further amount of test material (100 mg) was dispersed in 1 L of reconstituted water, with the pH adjusted to 11 using 1 M sodium hydroxide, to give a 100 mg/L stock dispersion. The pH of this dispersion was then adjusted to 9 using 1 M hydrochloric acid and any undissolved test material removed by filtration through a 0.2 µm Acrocap filter (first approximately 50 mL discarded to pre-condition the filter) to prepare the 100 % v/v saturated solution.
- Each stock dispersion and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: 1st instar
- Source: In-house laboratory cultures.
- Age: 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.
- Feeding during test: No

CULTURE CONDITIONS
- Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water adjusted to pH 9 in a temperature controlled room at approximately 20 °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.
- Daphnids were acclimatised to reconstituted water adjusted to pH 9 to ensure that they were not stressed during testing at pH 9. The daphnids were acclimatised for a period of 28 days to reconstituted water adjusted to pH 9 and during this period approximately 18 broods of young were produced.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg/L as CaCO3

Test temperature:

20.1 - 20.6 °C

pH:

8.0-9.0

Dissolved oxygen:

8.6 mg O2/L (95 - 97 % as a percentage of Air Saturation Value)

Nominal and measured concentrations:

Nominal: 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 % saturated solution

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass jars
- Type: The test vessels were covered to reduce evaporation
- Material, size, headspace, fill volume: Approximately 200 mL
- Aeration: No
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water: 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 9.0 ± 0.2 adjusted (if necessary) with NaOH or HCI and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
- Stock Solutions: a) CaCb·2H2O: 11.76 mg/L, b) MgSO4·7H2O: 4.93 mg/L, c) NaHCO3: 2.59 mg/L d) KCl: 0.23 mg/L
- The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.
- Culture medium different from test medium: The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals with the exception that a range-finding test was also conducted using reconstituted water without pH adjustment, at approximately pH 8.
- Intervals of water quality measurement: 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 the dissolved oxygen concentration were measured using a WTW pH/Oxi 3401 pH and dissolved oxygen meter and the temperature was measured using a Hanna Instruments HI93 510 digital thermometer.
- The reconstituted water for both the range-finding and definitive tests was the same as that used to maintain the stock animals with the exception that the range-finding test was also conducted using reconstituted water without pH adjustment, at approximately pH 8.

OTHER TEST CONDITIONS
- Adjustment of pH: The reconstituted water had a pH of 9.0 adjusted with NaOH or HCl.
- Photoperiod: The test vessels were maintained in the dark due to the suspected light sensitive nature of the test material

EFFECT PARAMETERS MEASURED: Any immobilisation or adverse reactions to exposure were recorded 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.

VEHICLE CONTROL PERFORMED: Yes. The control group was maintained under identical conditions but not exposed to the test material.

RANGE-FINDING STUDY
- Test concentrations: In the range-finding test Daphnia magna were exposed to a nominal test concentration of 100 % v/v saturated solution. The test material was prepared as a saturated solution from an initial dispersion of 100 mg/L using both pH 8 and pH 9 reconstituted water. The reconstituted water used for testing is normally approximately pH 8. However, the Sponsor indicated that the test material would be more soluble in reconstituted water adjusted to pH 9. Therefore, it was considered appropriate to conduct a range-finding test at both pH values to determine whether differences in toxicity would be observed.
- The daphnids were exposed to the concentrations prepared without filtration. Testing was conducted with and without filtration to determine whether physical effects of undissolved test material would occur.
- In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 21.1 °C to 21.6 °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 immobilised Daphnids were recorded. The control groups were maintained under identical conditions but not exposed to the test material.
- Results used to determine the conditions for the definitive study: Yes, based on the results of the range-finding test, the test material solutions for the definitive test were prepared by dispersing 100 mg/L of test material in pH adjusted reconstituted water. Any undissolved test material was removed by filtration to give a saturated solution of the test material. This 100 % v/v saturated solution was then further diluted, as necessary, to produce the remaining test concentrations of 1.0, 1.8, 3.2, 5.6, 10, 18, 32 and 56 % v/v saturated solution.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate at 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

27 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % confidence limits: 21 - 35 % v/v saturated solution

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 95 % confidence limits: 0.085 - 0.14 mg ai/L

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

5.6 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.022 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

SOLUBILITY TRIALS
- Initial solubility work indicated that the test material was more soluble in deionised reverse osmosis water than it was in reconstituted water by visual inspection and that a precipitate of the test material formed on its addition to reconstituted water. Therefore, a saturated solution of the test material was prepared to determine the amount of test material that could be dissolved.
- The results from analysis of the saturated solution prepared from an initial loading of 1000 mg/L showed that concentrations of test material were below the limit of quantitation of the analytical method.
- As the test material could not be detected in the initial saturated solution a second preparation was conducted in a similar manner to verify the findings of the initial analysis. The results from analysis again indicated that concentrations of the test material were below the limit of quantitation of the analytical method.
- Information provided by the Sponsor indicated that the test material would be more soluble at higher pH values and that at lower pH values the test material may change ionic form to a low water soluble protonated form. Therefore, it was considered justifiable to conduct further work including the range-finding test using reconstituted water adjusted to pH 9 in order to maximise the amount of dissolved test material for exposure.
- Given that a compound specific analytical method could not be developed without further extensive work it was considered appropriate to analyse the test concentrations during the definitive test using Total Organic Carbon (TOC) analysis as higher concentrations of test material could be detected using this method.

RANGE-FINDING TEST
- No immobilisation was observed at the test concentration of 100 % v/v saturated solution prepared using reconstituted water at pH 8 for either the filtered or unfiltered test media. However, 100 % immobilisation was observed at the 100 % v/v saturated solution prepared using reconstituted water at pH 9 for both the filtered and unfiltered test media.
- During the range-finding test the filtered test preparations were observed to be clear colourless solutions throughout the duration of testing. However, the unfiltered media were observed to be grey or grey/blue dispersions with the pH 9 media being darker in colour than the pH 8 media. Given that the test material was a dark blue solution, it was considered that the darker colouration of the pH 9 adjusted water indicated that a greater proportion of test material had dissolved when compared to the non-adjusted reconstituted water. Prior to adjustment from pH 11 to 9 the media was observed to be a dark blue solution, confirming information from the Sponsor that the test material showed a higher solubility at a higher pH. After 24 and 48 hours brown particles of undissolved test material were observed throughout the water column and settled to the bottom of the test vessels in the unfiltered media at both pH 8 and 9. Information provided by the Sponsor indicated that the test material may react with polyvalent cations in the reconstituted water such as calcium and magnesium. Therefore, it was considered that the precipitate observed may have been due to such a reaction. It was considered unlikely to be due to hydrolysis of the test material as information from the Sponsor indicated that the test material would not hydrolyse or form degradation products in the test medium. Given that a precipitate of the test material was observed in the unfiltered test media, it was considered appropriate to perform microscopic observations on the test animals.
- Microscopic inspection showed undissolved test material adhered to the antennae and thoracic appendages of the immobilised daphnids in the unfiltered test media. However, no undissolved test material was observed adhered to the immobilised daphnids in the filtered media. Given that immobilisation was shown in the unfiltered and filtered test media in reconstituted water adjusted to pH 9, it was considered appropriate to filter the test media during the definitive test to negate the possible physical effects of undissolved test material on the test animals.
- The difference shown in toxicity between the different pH media was considered to be due to more parent test material being dissolved in the media at a higher pH as information provided by the Sponsor indicated that the parent test material would be more soluble at higher pH values and that at lower pH values the test material changed ionic form to a low water soluble protonated form.
- Based on this information, the test material solutions for the definitive test were prepared by dispersing 100 mg/L of test material in pH adjusted reconstituted and then removing any undissolved test material by filtration. This 100 % v/v saturated solution was then further diluted, as necessary, to produce the remaining test concentrations of 1.0, 1.8, 3.2, 5.6, 10, 18, 32 and 56 % v/v saturated solution.

DEFINITIVE TEST
Immobilisation data:
- The 24 hour EC50 was 52 % v/v saturated solution (95 % confidence limits: 43-66 % v/v saturated solution) and the 48 hour EC50 was 27 % v/v saturated solution (95 % confidence limits: 21-35 % v/v saturated solution).
- The No Observed Effect Concentrations after 24 and 48 hours exposure were 10 and 5.6 % v/v saturated solution respectively. 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 3.9 (SE= 0.69) and 2.5 (SE= 0.34) respectively.

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

Physico-chemical measurements:
- Temperature was maintained at approximately 20 °C throughout the test. While there were no treatment related differences for oxygen concentration, concentration dependent differences in pH were observed throughout the test.

Total organic carbon analysis:
- During preliminary work a compound specific analytical method could not be developed for the test material. Therefore, it was considered appropriate to analyse the test concentrations during the definitive test using Total Organic Carbon (TOC) analysis as higher concentrations of test material could be detected using this method.
- Samples of the control and each test concentration were taken at 0 (fresh media) and 48 hours (old media) for Total Organic Carbon (TOC) analysis.
- The results from TOC analysis showed values of less than the control for the majority of the test concentrations except for the highest test level of 100 % v/v saturated solution which showed values of 0.233 and 0.354 mg C/L (corrected for the control values) at 0 and 48 hours respectively (0.315 and 0.480 mg/L as active ingredient). Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that all the results were around the limit of quantitation of the analytical method with the exception of the 100 % v/v test concentration. As the remaining test concentrations were all prepared from the 100 % v /v test concentration it was considered appropriate to calculate the amount of test material in each of the remaining test concentrations based on the mean measured TOC results shown for the 100 % v/v test concentration.
- Analysis of the immobilisation data by the probit method (Finney 1971) at 24 and 48 hours based on the mean measured TOC concentration of the 100 % v/v saturated solution gave the following results:
The 24 hour EC50 was 0.21 mg ai/L (95 % confidence limits: 0.17-0.26 mg ai/L) and the 48 hour EC50 was 0.11 mg ai/L (95 % confidence limits: 0.085-0.14 mg ai/L).
The No Observed Effect Concentrations after 24 and 48 hours exposure were 0.040 and 0.022 mg ai/L respectively. 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 3.8 (SE 0.67) and 2.5 (SE= 0.34) respectively.

Results with reference substance (positive control):

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 48-Hour EC50 value was 0.97 mg/L (95 % confidence limits: 0.85-1.1 mg/L).
The slope and standard error of the response curve at 24 hours was 9.8 (SE 2.2). Due to the unsuitable nature of the data it was not possible to calculate the slope and standard error of the response cuive at 48 hours.
The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour ECso value calculated from all positive controls was 0.84 mg/L (sd = 0.24).

Reported statistics and error estimates:

The EC50 value and associated confidence limits at 24 hours and the slope of the response curve and its standard error were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).
The EC50 value and associated confidence limits at 48 hours were calculated using the trimmed Spearman-Karber method (Hamilton et al., 1977) 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.

Table 1: Cumulative Immobilisation Data in the Definitive Test

Nominal Concentration (% v/v saturated solution)	Nominal Concentration (mg ai/L)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
		24 Hours				48 Hours
		R1	R2	Total	%	R1	R2	Total	%
Control	Control	0	0	0	0	0	0	0	0
1.0	0.0042	0	0	0	0	0	0	0	0
1.8	0.0072	0	0	0	0	0	0	0	0
3.2	0.013	0	0	0	0	0	0	0	0
5.6	0.022	0	0	0	0	0	0	0	0
10	0.040	0	0	0	0	3	2	5	25
18	0.072	0	2	2	10	4	4	8	40
32	0.13	2	2	4	20	5	5	10	50
56	0.22	2	4	6	30	6	7	13	65
100	0.40	10	10	20	100	10	10	20	100

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna was investigated and under the conditions of this study gave a 48-Hour EC50 value of 27 % v/v saturated solution with 95 % confidence limits of 21 – 35 % v/v saturated solution. The No Observed Effect Concentration at 48 hours was 5.6 % v/v saturated solution. The 48-Hour EC50 for the test material to Daphnia magna based on the mean measured TOC concentration of the 100 % v/v saturated solution was 0.11 mg ai/L with 95 % confidence limits of 0.085 - 0.14 mg ai/L. The No Observed Effect Concentration was 0.022 mg ai/L.

Executive summary:

The acute toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2., under GLP conditions in an acute immobilisation test with Daphnia magna.

Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test material at concentrations of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 % v/v saturated solution for 48 hours at a temperature of approximately 20 °C under static test conditions. The test material solutions were prepared by initially dispersing 100 mg/L of test material in pH adjusted reconstituted water prior to removing any undissolved test material by filtration to produce a saturated solution. A series of dilutions was made from this saturated solution to prepare the remainder of the test series. The number of immobilised Daphnia 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/1 for 48 hours at a temperature of approximately 20 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

The 48-Hour EC50 for the reference material to Daphnia magna based on nominal concentrations was 0.97 mg/L with 95 % confidence limits of 0.85 - 1.1 mg/L. The No Observed Effect Concentration was 0.56 mg/L.

During preliminary work a compound specific analytical method could not be developed for the test material. Therefore, it was considered appropriate to analyse the test concentrations during the definitive test using Total Organic Carbon (TOC) analysis as higher concentrations of test material could be detected using this method.

Samples of the control and each test concentration were taken at 0 (fresh media) and 48 hours (old media) for Total Organic Carbon (TOC) analysis.

The results from TOC analysis showed values of less than the control for the majority of the test concentrations except for the highest test level of 100 % v/v saturated solution which showed values of 0.233 and 0.354 mg C/L ( corrected for the control values) at O and 48 hours respectively (0.315 and 0.480 mg/L as active ingredient). Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that all the results were around the limit of quantitation of the analytical method with the exception of the 100 % v/v test concentration. As the remaining test concentrations were all prepared from the 100 % v/v test concentration it was considered appropriate to calculate the amount of test material in each of the remaining test concentrations based on the mean measured TOC results shown for the 100 % v/v test concentration.

The 48 h EC50 for the reference material bsed on nominal concentrations was 0.97 mg/L with 95 % confidence limits of 0.85 - 1.1 mg/L. The NOEC was 0.56 mg/L.

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna was investigated and under the conditions of this study gave a 48-Hour EC50 value of 27 % v/v saturated solution with 95 % confidence limits of 21 – 35 % v/v saturated solution. The No Observed Effect Concentration at 48 hours was 5.6 % v/v saturated solution.

The 48-Hour EC50 for the test material to Daphnia magna based on the mean measured TOC concentration of the 100 % v/v saturated solution was 0.11 mg ai/L with 95 % confidence limits of 0.085 - 0.14 mg ai/L. The No Observed Effect Concentration was 0.022 mg ai/L.

Description of key information

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna was investigated and under the conditions of this study gave a 48-Hour EC50 value of 27 % v/v saturated solution with 95 % confidence limits of 21 – 35 % v/v saturated solution. The No Observed Effect Concentration at 48 hours was 5.6 % v/v saturated solution.

The 48-Hour EC50 for the test material to Daphnia magna based on the mean measured TOC concentration of the 100 % v/v saturated solution was 0.11 mg ai/L with 95 % confidence limits of 0.085 - 0.14 mg ai/L. The No Observed Effect Concentration was 0.022 mg ai/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.11 mg/L

Additional information

The acute toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2., under GLP conditions in an acute immobilisation test with Daphnia magna. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test material at concentrations of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 % v/v saturated solution for 48 hours at a temperature of approximately 20 °C under static test conditions. The test material solutions were prepared by initially dispersing 100 mg/L of test material in pH adjusted reconstituted water prior to removing any undissolved test material by filtration to produce a saturated solution. A series of dilutions was made from this saturated solution to prepare the remainder of the test series. The number of immobilised Daphnia 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/1 for 48 hours at a temperature of approximately 20 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

The 48-Hour EC50 for the reference material to Daphnia magna based on nominal concentrations was 0.97 mg/L with 95 % confidence limits of 0.85 - 1.1 mg/L. The No Observed Effect Concentration was 0.56 mg/L.

During preliminary work a compound specific analytical method could not be developed for the test material. Therefore, it was considered appropriate to analyse the test concentrations during the definitive test using Total Organic Carbon (TOC) analysis as higher concentrations of test material could be detected using this method.

Samples of the control and each test concentration were taken at 0 (fresh media) and 48 hours (old media) for Total Organic Carbon (TOC) analysis.

The results from TOC analysis showed values of less than the control for the majority of the test concentrations except for the highest test level of 100 % v/v saturated solution which showed values of 0.233 and 0.354 mg C/L (corrected for the control values) at 0 and 48 hours respectively (0.315 and 0.480 mg/L as active ingredient). Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that all the results were around the limit of quantitation of the analytical method with the exception of the 100 % v/v test concentration. As the remaining test concentrations were all prepared from the 100 % v/v test concentration it was considered appropriate to calculate the amount of test material in each of the remaining test concentrations based on the mean measured TOC results shown for the 100 % v/v test concentration.

The 48 h EC50 for the reference material bsed on nominal concentrations was 0.97 mg/L with 95 % confidence limits of 0.85 - 1.1 mg/L. The NOEC was 0.56 mg/L.

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna was investigated and under the conditions of this study gave a 48-Hour EC50 value of 27 % v/v saturated solution with 95 % confidence limits of 21 – 35 % v/v saturated solution. The No Observed Effect Concentration at 48 hours was 5.6 % v/v saturated solution.

The 48-Hour EC50 for the test material to Daphnia magna based on the mean measured TOC concentration of the 100 % v/v saturated solution was 0.11 mg ai/L with 95 % confidence limits of 0.085 - 0.14 mg ai/L. The No Observed Effect Concentration was 0.022 mg ai/L.