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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 06 November 2012; Experimental Completion Date: 04 December 2012
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:
2013

Materials and methods

Test guidelineopen allclose all
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
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, 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 item in cases where the test item 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 item 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 item and water phase. At the completion of mixing and following a 1-Hour standing period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the
original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Test material form:
other: solid

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations:
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours.

- Sampling method:
Water samples were taken from the control and each loading rate WAF test group (replicates R1 – R4 pooled) at 0 and 48 hours for quantitative analysis.

- Sample storage conditions before analysis:
Samples were stored at approximately -20 °C prior to analysis.
Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.
Samples at the No Observed Effect Loading Rate and above only were analyzed.

Test solutions

Vehicle:
no
Details on test solutions:
TEST WATER:
Reconstituted water was used for both the range-finding and definitive test and is defined 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.

VALIDATION OF MIXING PERIOD:
Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.

A WAF of nominal loading rate of 100 mg/L was prepared, in duplicate, in demonized reverse osmosis water. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and the concentration of test item in the WAFs was verified by chemical analysis.

RANGE-FINDING TEST:
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the range-finding test the test item was prepared as a Water Accommodated Fraction (WAF).

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.

A sample of each loading rate WAF 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 concentrations within the range to be used for the definitive test were analyzed.

DEFINITIVE TEST:
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Experimental Preparation:
Amounts of test item (20, 64, 20 ,64 and 200 mg) were each separately added to the surface of 20, 20, 2, 2 and 2 liters of reconstituted water to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively. After the addition of the test item, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the 32 and 100 mg/L water columns and hence it was considered justifiable to remove these WAFs by filtering through a glass wool plug (2-4 cm in length).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. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 32 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed that the glass wool plug had removed all of the dispersed test item in the 32 mg/L loading rate WAF, however, filtration through postlip filter paper was needed to remove all of the dispersed test item in the 100 mg/L loading rate WAF. No micro particles of test item were observed in the 1.0, 3.2 and 10 mg/L loading rate WAF and so they were removed as above but without the use of glass wool or filter paper.
























Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
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 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 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.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
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 20°C to 21ºC throughout the test
pH:
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
pH was recorded at the start and termination of the test.
pH in test vessels ranged from 7.8 - 8.0
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 concentration was recorded at the start and termination of the test.
There were no treatment related differences for oxygen concentration.
Nominal and measured concentrations:
Definitive test:
Nominal concentrations: 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF.
Measured: Near nominal measured concentrations were observed in the 10 to 100 mg/L loading rates.
Details on test conditions:
EXPOSURE CONDITIONS:
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 20°C to 21°C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods with a light intensity ranging from 603 to 694 lux. 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 immobilization 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 immobilized if they were unable to swim for approximately 15 seconds after gentle agitation.

VORTEX DEPTH MEASUREMENTS:
The vortex depth was recorded at the start and end of the mixing period.








Reference substance (positive control):
yes
Remarks:
potassium dichromate.

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
20 other: mg/L Loading Rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilization
Remarks on result:
other: 48 h 95% Confidence Limits: 15-29 mg/L Loading Rate WAF
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
3.2 other: mg/L Loading Rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilization
Details on results:
VALIDATION OF MIXING PERIOD:
Pre-study investigational work indicated that there was no significant increase in the measured concentration by extending the preparation period for longer than 24 hours. Therefore, for the purpose of testing the test item was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

RANGE-FINDING TEST:
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1 (see attached background material)

No immobilization was observed at 1.0 mg/L loading rate WAF. However, immobilization was observed at 10 and 100 mg/L loading rate WAF.

Based on this information loading rates of 1.0, 3.2, 10, 32 and 100 mg/L, using a stirring period of 23 hours followed by a 1-Hour standing period, were selected for the definitive test.

Chemical analysis of the 1.0 and 100 mg/L loading rate WAF preparations at 0 hours (see Appendix 6; Analytical Investigations, attached background material) showed measured concentrations of 1.06 and 70.4 mg/L respectively were obtained. Analysis of the test preparations at 48 hours showed measured test concentrations of 1.11 and 70.3 mg/L were obtained from the 1.0 and 100 mg/L loading rate WAF preparations respectively indicating that the test item was stable over the test duration.

DEFINITIVE TEST:
Immobilization Data:
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2. The relationship between percentage immobilization and concentration at 24 and 48 hours is given in Figure 1 and Figure 2 (see attached background material).

Analysis of the immobilization data by the trimmed Spearman-Karber method at 24 and 48 hours based on the nominal loading rates gave the following results:
24 h EL50 (mg/L Loading Rate WAF): 92 (95% Confidence limits 68 - 158 mg/L Loading Rate WAF)
48 h EL50 (mg/L Loading Rate WAF): 20 (95% Confidence limits 15 - 29 mg/L Loading Rate WAF)

The No Observed Effect Loading rates after 24 and 48 hours exposure were 10 and 3.2 mg/L loading rate WAF respectively. Correspondingly the Lowest Observed Effect loading rates at 24 and 48 hours were 32 and 10 mg/L loading rate WAF respectively.

PHISCO-CHEMICAL MEASUREMENTS:
Temperature was maintained at 20°C to 21°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

VORTEX DEPTH MEASUREMENTS:
The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion (see Table 3 - attached background material).

OBSERVATIONS ON TEST ITEM SOLUBILITY:
Observations on the test media were carried out during the mixing and testing of the WAFs.

At the start of the mixing period the 1.0, 3.2, 10, 32 and 100 mg/L loading rates were observed to be clear colorless water columns with test item on the base of the vessel. After 23 hours stirring and a 1-Hour standing period the 1.0, 3.2, and 10 mg/L loading rates were observed to remain as clear colorless water column with test item visible on the base of the vessel. However, the 32 and 100 mg/L loading rates were observed to be cloudy water column with test item on the base of the vessel. Microscopic examination of the WAFs showed that particles of test item were visible in the 32 and 100 mg/L loading rate WAFs and therefore it was considered justifiable to remove the 32 and 100 mg/L loading rate WAFs by filtering through a glass wool plug (2-4 cm in length). Microscopic examination after filtering showed that the glass wool plug had removed all of the dispersed test item in the 32 mg/L loading rate WAF, however, filtration through postlip filter paper was needed to remove all of the dispersed test item in the 100 mg/L loading rate WAF. During the test the 1.0, 3.2, 10, 32 and 100 mg/L loading rates were observed to be clear, colorless solutions.

CHEMICAL ANALYSIS OF TEST LOADING RATES:
A lower than expected measured test concentration was obtained in the 3.2 mg/L loading rate WAF, and this may be due to the inevitable variability that occurs when working with such small amounts of test item. This was not considered to have had an effect on the validity of the study given that all results were based on nominal loading rates.

Although the substituted-(naphthalen-1-methyl)heteromonocyclic chloride content of the test item was determined to be highly water soluble and propane-1,2-diol is known to be miscible with water, the preparation of WAFs was considered the most suitable method of test solution preparation. This was due to the presence of lesser soluble/insoluble impurities within the test item.

After stirring, and following a 1-Hour standing period, dispersed test item was observed to be present in the 32 and 100 mg/L loading rate WAFs and hence it was considered appropriate to filter the WAF through a glass wool plug and/or filter paper. Observations made after filtration indicated that no dispersed test item remained. However, following filtration and analysis, the analyzed concentration approximated to the nominal test item loading rate. This was as expected, as analysis was based on a single dominant peak monitored in the UV region by HPLC, attributed to the substituted-(naphthalen-1-methyl)heteromonocyclic chloride content of the test item. Therefore, as quantification was based on standard solutions prepared from the original test item and monitoring of this same, single peak, complete aqueous dissolution/extraction of the substituted-(naphthalen-1-methyl)heteromonocyclic chloride content of the test item was supported. However filtration prior to exposure remained essential to remove the undissolved test item originating as a result of the impurities present.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.







Results with reference substance (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 postive control were similar to those in the definitive test.

Analysis of the immobilization data by the geometric mean method at 24 hours and the trimmed Spearman-Karber method at 48 hours based on the nominal test concentrations gave the following results@

24 h EC50: 0.75 mg/L
48 h EC50: 0.45 mg/L

The No Observed Effect Concentrations after 24 and 48 hours were 0.56 and 0.32 mg/L respectively. The No Observed Effect Concentration is based upon zero immobilization at this concentration.

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


Any other information on results incl. tables

Results of chemical analysis gave the following results:

 

Nominal Loading Rate WAF

0 Hour

48 Hour

Measured Concentration (mg/L)

% of nominal

Measured Concentration (mg/L

% of nominal

Control

<LOQ

-

<LOQ

-

3.2

1.75/1.75**

55/55**

1.73/1.98**

54/62**

10

11.1

111

10.9

109

32

31.5

98

31.0

97

100

101

101

100

100

 ** Duplicate sample stored at approximately -20°C prior to analysis.

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 EL50 value of 20 mg/L loading rate WAF with 95% confidence limits of 15 - 29 mg/L loading rate WAF. The Lowest Observed Effect Loading rate was considered to be 10 mg/L loading rate WAF. The No Observed Effect Loading rate was 3.2 mg/L loading rate WAF.
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

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test item was prepared as a Water Accommodated Fraction (WAF).

 

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L for 48 hours at a temperature of 20°C to 21°C under static test conditions. The number of immobilized Daphnia 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 loading rates was 20 mg/L loading rate WAF with 95% confidence limits of 15 - 29 mg/L loading rate WAF. The Lowest Observed Effect Loading rate was considered to be 10 mg/L loading rate WAF. The No Observed Effect Loading rate was 3.2 mg/L loading rate WAF.

Chemical analysis of the test preparations gave the following results

Nominal Loading Rate WAF

0 Hour

48 Hour

Measured Concentration (mg/L)

% of nominal

Measured Concentration (mg/L

% of nominal

Control

<LOQ

-

<LOQ

-

3.2

1.75/1.75**

55/55**

1.73/1.98**

54/62**

10

11.1

111

10.9

109

32

31.5

98

31.0

97

100

101

101

100

100

** Duplicate sample.

Although the substituted-(naphthalen-1-methyl)heteromonocyclic chloride content of the test item was determined to be highly water soluble and propane-1,2-diol is known to be miscible with water, the preparation of WAFs was considered the most suitable method of test solution preparation. This was due to the presence of lesser soluble/insoluble impurities within the test item.

After stirring, and following a 1-Hour standing period, dispersed test item was observed to be present in the 32 and 100 mg/L loading rate WAFs and hence it was considered appropriate to filter the WAF through a glass wool plug and/or filter paper. Observations made after filtration indicated that no dispersed test item remained. However, following filtration and analysis, the analyzed concentration approximated to the nominal test item loading rate. This was as expected, as analysis was based on a single dominant peak monitored in the UV region by HPLC, attributed to the substituted-(naphthalen-1-methyl)heteromonocyclic chloride content of the test item. Therefore, as quantification was based on standard solutions prepared from the original test item and monitoring of this same, single peak, complete aqueous dissolution/extraction of the substituted-(naphthalen-1-methyl)heteromonocyclic chloride content of the test item was supported. However filtration prior to exposure remained essential to remove the undissolved test item originating as a result of the impurities present.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.