2-Propenoic acid, butyl ester, reaction products with butadiene, sulfur and tri-Ph phosphite

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:

7 July 2009 - 20 August 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, without deviations from standard test guidelines

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

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-Propenoic acid, butyl ester, reaction products with butadiene, sulfur and tri-Ph phosphite

EC Number:

300-339-7

EC Name:

2-Propenoic acid, butyl ester, reaction products with butadiene, sulfur and tri-Ph phosphite

Cas Number:

93925-37-2

Molecular formula:

C18H15O3P.C7H12O2.C4H6.S as an example

IUPAC Name:

2-Propenoic acid, butyl ester, reaction products with butadiene, sulfur and tri-Ph phosphite

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l

- Sampling method: Water samples were taken from the control (replicates R1 – R2 pooled) and each loading rate WAF test group (replicates R1 – R2 pooled) at 0 (fresh media), 24 (old and fresh media) and 48 (old media) hours for quantitative analysis.

- Sample storage conditions before analysis: Samples were used immediately. Duplicate samples were taken and stored at -20 C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accommodated Fractions (WAFs) solution preparation. The aqueous phase or WAF was removed by mid-depth siphoning method.

- Eluate: Not applicable.

- Differential loading: Water Accommodated Fractions (WAFs) loading rate.

- Controls: Untreated negative control and positive control which conducted every 6 months.

- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water Flea

- Strain: Daphnia magna

- Source: In house laboratory cultures

- Age at study initiation (mean and range, SD): 1st instar, less than 24 h old

- Weight at study initiation (mean and range, SD): no data available.

- Length at study initiation (length definition, mean, range and SD): no data available.

- Valve height at study initiation, for shell deposition study (mean and range, SD): no data available.

- Peripheral shell growth removed prior to test initiation: no data available.

- Method of breeding: parthenogenises

- Feeding during test : The daphnids received no food during exposure
- Food type: Not applicable.
- Amount: Not applicable.
- Frequency: Not applicable.

ACCLIMATION
- Acclimation period: Not applicable.

- Acclimation conditions (same as test or not): Not applicable.

- Type and amount of food: Each culture was fed daily with a suspension of algae (Chlorella sp.).

- Feeding frequency: daily

- Health during acclimation (any mortality observed): Not applicable.


QUARANTINE (wild caught)
- Duration: Not applicable.
- Health/mortality: Not applicable.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

The number of immobilised Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

Test conditions

Hardness:

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

Test temperature:

21ºC to 22ºC

pH:

There were no treatment related differences in pH (pH = 8.0).

Dissolved oxygen:

There were no treatment related differences in oxygen concentration (%ASV = 98~102%).

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal loading rates of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56, and 100 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass jars
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 ml glass jars containing approximately 250 ml of test preparation were used.
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): the test media renewal at 24 hours.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): Duplicate test vessels were used for each test and control group
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): Not applicable.
- Biomass loading rate: No data available.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water
- Total organic carbon: NDA
- Particulate matter: NDA
- Metals: NDA
- Pesticides: NDA
- Chlorine: NDA
- Alkalinity: NDA
- Ca/mg ratio: NDA
- Conductivity: NDA
- Culture medium different from test medium: No difference
- Intervals of water quality measurement: N/A

OTHER TEST CONDITIONS
- Adjustment of pH: Using NaOH or HCl were necessary

- Photoperiod: 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods

- Light intensity: NDA

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8x

- Justification for using less concentrations than requested by guideline: NA

- RANGE FINDING STUDY
- Test concentrations: nominal loading rates of 1.0, 10 and 100 mg/l

- Results used to determine the conditions for the definitive study: No immobilisation was observed at the 1.0 mg/l loading rate WAF. However, immobilisation was observed at the 10 and 100 mg/l loading rate WAFs.

Based on this information loading rates of 1.0, 1.8, 3.2, 5.6, 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.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (non-concurrent)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 8.4 - 13 mg/L WAF

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: NDA
- Observations on body length and weight: NDA
- Other biological observations: NDA
- Mortality of control: 0
- Other adverse effects control: None noted
- Abnormal responses: NDA
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None noted
- Effect concentrations exceeding solubility of substance in test medium: N/A

Results with reference substance (positive control):

- Results with reference substance valid? Yes.

- Mortality: mortality was observed at concentration ≥ 0.56 mg/L

- EC50/LC50:
Time (h) 3

EC50 (mg/l) > 3.2
95% Confidence limits (mg/l) - no data

Time (h) 24
EC50 (mg/l) = 1.0
95% Confidence limits (mg/l) = 0.90 – 1.2

Time (h) 48
EC50 (mg/l) = 0.78
95% Confidence limits (mg/l) = 0.68 - 0.88

Reported statistics and error estimates:

No data available

Any other information on results incl. tables

Cumulative Immobilisation data in the Range Finding Test

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	2
100	0	4

Cumulative Immobilisation Data in the Definitive Test

Nominal Loading Rate (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0	0
1.8	0	0	0	0	0	0	0	0
3.2	0	0	0	0	0	2	2	10
5.6	0	0	0	0	3	3	6	30
10	0	0	0	0	5	4	9	45
18	0	0	0	0	6	7	13	65
32	0	0	0	0	9	9	18	90
56	0	0	0	0	10	10	20	100
100	10	10	20	100	10	10	20	100

Chemical analysis of test loading rates

Chemical analysis of the test preparations (see Appendix 3) at 0 (fresh media), 24 (old and fresh media) and 48 (old media) hours showed measured test concentrations to range from 0.015 mg/l to 0.265 mg/l. A concentration dependant general trend was consistently observed (with increasing loading rate) in both the fresh and old media at both media renewal periods, and a slight decline in measured concentration was consistently observed over each media renewal period.

The control test sample at 0 hours showed a measured concentration of 0.0030 mg/l. Analysis of a frozen duplicate sample showed a measured concentration of less than the limit of quantification (LOQ) which confirmed the original analysis to be erroneous.

The results of the chemical analysis showed measured concentrations in the definitive test that were lower than the measured Total Organic Carbon (TOC) levels observed in the pre-study validation of mixing period work. This was considered to be due to the analytical method employed only detecting the 399 ion whereas TOC analysis detected the total dissolved test material present in the WAF.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*50 value of 11 mg/l loading rate WAF with 95% confidence limits of 8.4 - 13 mg/l loading rate WAF. The No Observed Effect Loading rate at 48 hours was 1.8 mg/l loading rate WAF.

Executive summary:

Introduction.A study was performed to assess the acute toxicity of the test material to daphnia magna followed that described in the OECD 202.

Methods.Following a preliminary range-finding test, twenty daphnids (2 replicates of

10 animals) were exposed to Water Accommodated Fractions (WAFs) of the test material over a range of nominal loading rates of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l for 48 hours at a temperature of 21ºC to 22ºC under semi-static test conditions. The number of immobilised Daphnia and any adverse reactions to exposure 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 under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results.The 48-Hour EL*50 for the test material to Daphnia magna based on nominal loading rates was 11 mg/l loading rate WAF with 95% confidence limits of 8.4 - 13 mg/l loading rate WAF. The No Observed Effect Loading rate was 1.8 mg/l loading rate WAF.

Chemical analysis of the test preparations at 0 (fresh media), 24 (old and fresh media) and 48 (old media) hours showed measured test concentrations to range from 0.015 mg/l to 0.265 mg/l. A concentration dependant general trend was consistently observed (with increasing loading rate) in both the fresh and old media at both media renewal periods, and a slight decline in measured concentration was consistently observed over each media renewal period.

The control test sample at 0 hours showed a measured concentration of 0.0030 mg/l.

Analysis of a frozen duplicate sample showed a measured concentration of less than the limit of quantification (LOQ) which confirmed the original analysis to be erroneous.

The dissolved test material may have been one or several components of the test material. Given that toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole, the results were based on nominal loading rates only.

The 48-Hour EC50 for 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.