Registration Dossier

Ecotoxicological information

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:
18 January 2010 and 19 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Validation of mixing period
Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances in the WAF. A WAF of nominal loading rate of 100 mg/l was prepared, in duplicate, in culture medium. 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 samples taken for Total Organic Carbon analysis (see Appendix 3 in any other information on materials and methods section).

Test solutions

Vehicle:
no
Details on test solutions:
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.
The test was conducted in 250 ml glass conical flasks each completely filled with test preparation and sealed with a ground glass stopper to reduce evaporation and minimise losses due to the test item’s volatile nature. Two replicate flasks were used for each control and test concentration.
Amounts of test item (22.5, 225 and 2250 mg) were each separately added to the surface of 22.5 litres of reconstituted water to give the 1.0, 10 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. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 1.0, 10 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 20ºC 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 300 ml glass conical flask was completely filled with test preparation and sealed with a ground glass stopper to reduce evaporation and minimise losses due to the test items volatile nature. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.
The control group was maintained under identical conditions but not exposed to the test item.

Definitive test
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/l.

Experimental preparation
Amounts of test item (110, 198, 352, 616 and 1100 mg) were each separately added to the surface of 11 litres of reconstituted water to give the 10, 18, 32, 56 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. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 10, 18, 32, 56 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours (see Appendix 2 in attached section).

Physico-chemical measurements
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 was measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period.

Positive Control
A positive control (Harlan Laboratories Ltd Project No: 0039/1133) conducted approximately every six months used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
An amount of reference item (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 approximately 20°C




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:
Not stated


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


- 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.


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.

QUARANTINE (wild caught)

- Duration:
Not applicable

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 21 deg C to 22 deg 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 in any other information on materials and methods section.
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 in any other information on materials and methods section.
Salinity:

freshwater used.
Nominal and measured concentrations:
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.
Details on test conditions:
TEST SYSTEM
- Test vessel:
Exposure conditions
As in the range-finding test 300 ml completely filled ground glass stoppered conical flasks containing approximately 300 ml of test preparation were used. At the start of the test 10 daphnids were placed in each test and control vessel at random, in the test preparations. Duplicate test vessels were used for each test and control group. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21ºC to 22ºC 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. 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.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 95% CL of 30-41 mg/l.
Duration:
48 h
Dose descriptor:
other: NOEL
Effect conc.:
10 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:
Validation of Mixing Period
Pre-study work (see Appendix 3 see in any other information on materials and methods section) indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 hours. As such, for the purposes of testing, the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour standing period prior to the removal of the aqueous phase by mid-depth siphon.

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 (see in any other information on results section).
No immobilisation was observed at 1.0 and 10 mg/l loading rate WAF. However, 100% immobilisation was observed at the 100 mg/l loading rate WAF.
Based on this information loading rates of 10, 18, 32, 56 and 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, were selected for the definitive test.

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 (see in any other information on results section). The relationship between percentage immobilisation and loading rate at 24 and 48 hours is given in Figures 1 and 2 (see in attached section).
Analysis of the immobilisation data by the probit method (Finney 1971) at 24 and 48 hours based on the nominal loading rates gave the following results:
Time (h) EL*50 (mg/l Loading Rate WAF) 95% Confidence limits
(mg/l Loading Rate WAF)
24 49 41 - 60
48 35 30 - 41
The No Observed Effect Loading rate after 24 and 48 hours exposure was 10 mg/l loading rate WAF. The No Observed Effect Loading rate is based upon zero immobilisation at this loading rate.
The slopes and their standard errors of response curves at 24 and 48 hours were 4.6(SE = 0.8) and 6.0(SE = 1.1) respectively.

Physico-chemical measurements
The results of the physico-chemical measurements are given in Appendix 4 see in any other information on materials and methods section. Temperature was maintained at 21ºC to 22ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Some of the temperatures during the test were 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 no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperature range was within guideline specification.

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 4 in anu other information on results section.

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 all loading rates were observed to be clear, colourless water columns with oily globules of test item floating at the surface. After 23 hours stirring and a 1-Hour standing period the loading rates were observed to be clear, colourless water columns with an oily slick of test item at the surface. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, all the loading rates were observed to be clear, colourless solutions.

Chemical analysis of test loading rates
Analysis of the test preparations at 0 hours (see Appendix 2) showed measured test concentrations to range from 3.91 to 37.6 mg/l. A decline in measured test concentrations was observed at 48 hours in the range of 3.06 to 30.5 mg/l.
Given that these results ranged from 78% to 81% of the 0-Hour measured test concentrations it was considered that the decline observed in the vessels which had been opened on a daily basis was due mainly to losses through volatility.
However, 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):
Positive Control
Cumulative immobilisation data from the exposure of Daphnia magna to the reference item (Harlan Laboratories Ltd Project No: 0039/1133) during the positive control are given in Table 3 (see in any other information on results section). The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 3 and 4 see in attached section.
Analysis of the immobilisation data by the probit method (Finney 1971) at 24 hours and the trimmed Spearman-Karber method (Hamilton et al 1977) at 48 hours based on the nominal test concentrations gave the following results:
Time (h)
EC50 (mg/l) 95% Confidence limits
(mg/l)
24 0.84 0.72 - 0.97
48 0.65 0.58 - 0.72
The No Observed Effect Concentrations after 24 and 48 hours was 0.32 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
The slope and its standard error of the response curve at 24 hours was 7.7 (SE = 1.6). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of response curve at 48 hours.
The results from the positive control with potassium dichromate were within the normal range for this reference item. The mean 48-Hour EC50 value calculated from all positive controls was 0.77 mg/l (sd = 0.20).
Reported statistics and error estimates:
The EL*50 values and associated confidence limits at 24 and 48 hours 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.
The EC50 value and associated confidence limits at 24 hours and the slope of the response curve and 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 by the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999).
Probit analysis is used when 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

Table1              Cumulative Immobilisation Data in the Range-findingTest

Nominal Loading Rate

(mg/l)

Cumulative ImmobilisedDaphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Control

0

0

1.0

0

0

10

0

0

100

10

10

Table2              Cumulative Immobilisation Data in the DefinitiveTest

Nominal Loading Rate

(mg/l)

Cumulative ImmobilisedDaphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

R1

R2

Total

%

R1

R2

Total

%

Control

0

0

0

0

0

0

0

0

10

0

0

0

0

0

0

0

0

18

1

0

1

5

1

1

2

10

32

1

1

2

10

1

4

5

25

56

6

8

14

70

10

9

19

95

100

9

9

18

90

10

10

20

100



R1– R2= Replicates 1 and 2

Table3              Cumulative Immobilisation Data in the Positive Control

Nominal
Concentration
(mg/l)

Cumulative ImmobilisedDaphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

R1

R2

Total

%

R1

R2

Total

%

Control

0

0

0

0

0

0

0

0

0.32

0

0

0

0

0

0

0

0

0.56

1

1

2

10

3

2

5

25

1.0

7

7

14

70

10

10

20

100

1.8

10

10

20

100

10

10

20

100

3.2

10

10

20

100

10

10

20

100

Table4              Vortex Depth Measurements at the Start and End of the Mixing Period

 

Nominal Loading Rate (mg/l)

Control

10

18

*

+

*

+

*

+

Height of Water Column (cm)

28.0

28.0

28.0

28.0

28.0

28.0

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

 

 

Nominal Loading Rate (mg/l)

32

56

100

*

+

*

+

*

+

Height of Water Column (cm)

28.0

28.0

28.0

28.0

28.0

28.0

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

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 item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*50 value of 35 mg/l loading rate WAF with 95% confidence limits of 30 41 mg/l loading rate WAF. The No Observed Effect Loading rate at 48 hours was 10 mg/l loading rate WAF.
Executive summary:

Introduction.A study was performed to assess the acute toxicity of the test item 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 Regulation (EC) No. 440/2008.

Methods.Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 10, 18, 32, 56 and 100 mg/l for 48 hours at a temperature of 21°C to 22ºC under static test conditions. The number of immobilisedDaphniawere recorded after 24 and 48 hours.

The test item was known to be volatile and hence testing was conducted in completely filled, stoppered test vessels in order to minimise possible losses due to volatilisation.

A positive control conducted approximately every six months used potassium dichromate as the reference item. Daphnia magnawas exposed to an aqueous solution of the reference item 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. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results.The 48-Hour EL*50for the test item toDaphnia magnabased on nominal loading rates was 35 mg/l loading rate WAF with 95% confidence limits of 30 - 41 mg/l loading rate WAF. The No Observed Effect Loading rate was 10 mg/l loading rate WAF.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 3.91 to 37.6 mg/l. A decline in measured test concentrations was observed at 48 hours in the range of 3.06 to 30.5 mg/l.

Given that these results ranged from 78 – 81% of the 0-Hour measured test concentrations it was considered that the decline observed in the vessels which had been opened on a daily basis was due mainly to losses through volatility. 

However, 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.

The 48-Hour EC50for the reference item toDaphnia magnabased on nominal concentrations was 0.65 mg/l with 95% confidence limits of 0.58 – 0.72 mg/l. The No Observed Effect Concentration was 0.32 mg/l.


*EL = Effective Loading rate

 CONCLUSION

The acute toxicity of the test item to the freshwater invertebrateDaphnia magnahas been investigated and gave a 48-Hour EL*50value of 35 mg/l loading rate WAF with 95% confidence limits of 30 - 41 mg/l loading rate WAF. The No Observed Effect Loading rate at 48 hours was 10 mg/l loading rate WAF.


*EL = Effective Loading rate