Cyclohexane-1,3-dione

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:

9 December 2009 and 30 December 2009.

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP inspection: 15 September 2009 Date of Signature on GLP certificate: 26 November 2009

Test material

Test material information

Constituent 1

Reference substance name:

1,3-Cyclohexanedione

IUPAC Name:

1,3-Cyclohexanedione

Details on test material:

Sponsor's identification :1,3-Cyclohexanedione
Description :pale beige solid
Batch number :1091224
Date received :24 November 2009
Storage conditions :approximately 4°C in the dark
The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor. A Certificate of Analysis for the test material supplied by the Sponsor is given in Appendix 1 (see in attached section).

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Verification of test concentrations
Water samples were taken from the control and each test group (replicates R1 - R2 pooled) at 0 and 48 hours for quantitative analysis.
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
The method of analysis, stability, recovery and test preparation analyses are described in attached Appendix 3.

Test solutions

Vehicle:

no

Details on test solutions:

Range-finding test
The test concentrations 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 test concentrations of 0.10, 1.0, 10 and 100 mg/l. The test material was dissolved directly in water.
An amount of test material (50 mg) was dissolved in reconstituted water with the aid of ultrasonication for approximately 3 minutes and the volume adjusted to 500 ml to give a 100 mg/l test concentration. A series of dilutions was made from the 100 mg/l test concentration to give further test concentrations of 10, 1.0 and 0.10 mg/l.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 20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 Daphnia magna were recorded.
The control group was maintained under identical conditions but not exposed to the test material.

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

Experimental preparation
For the purpose of the definitive test the test material was dissolved directly in water.
An amount of test material (200 mg) was dissolved in reconstituted water with the aid of ultrasonication for approximately 3 minutes and the volume adjusted to 2 litres to give a 100 mg/l test concentration. A series of dilutions was made from the 100 mg/l test concentration to give further test concentrations of 56, 32, 18 and 10 mg/l.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 48 hours (see Appendix 3).

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 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 approximately 20°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 material.
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.

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.
These young were removed from the cultures and used for testing.

- 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.
The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods

- 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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

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

Test temperature:

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:

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l. The test material was dissolved directly in water.
An amount of test material (50 mg) was dissolved in reconstituted water with the aid of ultrasonication for approximately 3 minutes and the volume adjusted to 500 ml to give a 100 mg/l test concentration. A series of dilutions was made from the 100 mg/l test concentration to give further test concentrations of 10, 1.0 and 0.10 mg/l.

Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 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 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 approximately 20°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 material.
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.

The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.
The reconstituted water is defined in the Attached Appendix 2.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL of 23 - 24 mg/l.

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

18 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 material during the range-finding test are given in Table 1.
No immobilisation was observed at the test concentrations of 0.10, 1.0 and 10 mg/l. However, immobilisation was observed at 100 mg/l.
Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/l were selected for the definitive test.

Definitive Test

Immobilisation data
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 1 and 2.
Based on the nominal test concentrations 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 gave the following results:
Time (h) EC50 (mg/l) 95% Confidence limits
(mg/l)
24 93 66 - 180
48 24 23 - 24
The No Observed Effect Concentration after 24 and 48 hours exposure was 18 mg/l. The No Observed Effect Concentration is based upon no significant immobilisation at this concentration.
The slope and standard error of the response curve at 24 hours was 2.5 (SE = 0.59). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of response curve at 48 hours.

Observations on test material solubility
At the start and throughout the duration of the test all test concentrations were observed to be clear colourless solutions.

Physico-chemical measurements
The results of the physico-chemical measurements are given in Appendix 4. Temperature was maintained at approximately 20C throughout the test. Whilst there were no treatment related differences for oxygen concentration a concentration dependant decline in pH was observed. Given that this was considered to be due to an intrinsic property of the test material no attempt was made to alter the pH prior to exposure. Inspection of the immobilisation data showed that immobilisation only occurred where the pH of the test vessels was significantly lower than that of the control vessels which would suggest that the acidic nature of the test material may have contributed towards to toxicity observed.

Verification of test concentrations
Analysis of the test preparations at 0 hours (see Appendix 3) showed that whilst a near nominal concentration was obtained from the 100 mg/l test sample, concentrations in the range of 10% to 72% of nominal were obtained from the 10, 18, 32 and 56 mg/l test samples. Given that analysis of the test samples at 48 hours showed near nominal concentrations were obtained from all test samples it was considered appropriate to analyse duplicate 0-Hour test samples which had been stored frozen. Analysis of these duplicate samples showed near nominal concentrations were obtained which would suggest that the original results obtained were erroneous. As such it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations only.

Positive Control
Cumulative immobilisation data from the exposure of Daphnia magna to the reference material (Harlan Laboratories Ltd Project No: 0039/1094) during the positive control are given in Table 3. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 3 and 4.
Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the probit method (Finney 1971) at 24 and 48 hours based on the nominal test concentrations gave the following results:
Time (h) EC50 (mg/l) 95% Confidence limits (mg/l)

3 > 3.2 -
24 1.0 0.90 - 1.2
48 0.78 0.68 - 0.88

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 immobilisation at this concentration.
The slopes and their standard errors of the response curves at 24 and 48 hours were 7.8 (SE = 1.7) and 12 (SE = 2.4) respectively.
The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/l (sd = 0.20).

Results with reference substance (positive control):

Positive Control
Cumulative immobilisation data from the exposure of Daphnia magna to the reference material (Harlan Laboratories Ltd Project No: 0039/1094) 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).
Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the probit method (Finney 1971) at 24 and 48 hours based on the nominal test concentrations gave the following results:
Time (h) EC50 (mg/l) 95% Confidence limits (mg/l)

3 > 3.2 -
24 1.0 0.90 - 1.2
48 0.78 0.68 - 0.88

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 immobilisation at this concentration.
The slopes and their standard errors of the response curves at 24 and 48 hours were 7.8 (SE = 1.7) and 12 (SE = 2.4) respectively.
The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/l (sd = 0.20).

Reported statistics and error estimates:

The EC50 values 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) and at 48 hours by the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999).

Any other information on results incl. tables

Table1              Cumulative Immobilisation Data in the Range-finding Test

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
0.10	0	0
1.0	0	0
10	0	0
100	2	8

 

Table2              Cumulative Immobilisation Data in the Definitive Test

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
10	0	1	1*	5	0	1	1*	5
18	0	0	0	0	0	0	0	0
32	1	1	2	10	10	10	20	100
56	2	3	5	25	10	10	20	100
100	6	6	12	60	10	10	20	100

 

Table 3              Cumulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	3 Hours				24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0	0	0	0	0
0.56	0	0	0	0	0	0	0	0	1	0	1	5
1.0	0	0	0	0	7	3	10	50	10	8	18	90
1.8	0	0	0	0	10	9	19	95	10	10	20	100
3.2	0	0	0	0	10	10	20	100	10	10	20	100

R₁– R₂= Replicates 1 and 2

*Immobilisation observed at 10 mg/l was considered to be due to natural causes rather than true toxicity because the observed effect was less than 10% and no further immobilisation was observed.

R₁– R₂= Replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The required quality criteria (validity, reliability, repeatability) have been fulfilled within this document.

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of 24 mg/l; 95% confidence limits 23 24 mg/l. The No Observed Effect Concentration at 48 hours was 18 mg/l.

Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in 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 (2 replicates of 10 animals) were exposed to an aqueous solution of the test material at concentrations of 10, 18, 32, 56 and 100 mg/l for 48 hours at a temperature of approximately 20°C under static test conditions. 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/l for 48 hours at a temperature of 21°C to 22°C understatic test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results.

The 48-Hour EC₅₀ for the test material to Daphnia magna based on nominal test concentrations was 24 mg/l; 95% confidence limits 23 - 24 mg/l. The No Observed Effect Concentration was 18 mg/l.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

The 48-Hour EC₅₀for the reference material to Daphnia magna based on nominal concentrations was 0.78 mg/l with 95% confidence limits of 0.68 – 0.88 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

Conclusion

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC₅₀value of 24 mg/l; 95% confidence limits  23 ‑ 24 mg/l. The No Observed Effect Concentration at 48 hours was 18 mg/l.