Pentaerythritol tetrakis(3-mercaptopropionate)

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:

26 January - 11 March 2009

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)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Analytical monitoring:

yes

Details on sampling:

The concentration, homogeneity and stability of the test material in the test perparations were verified by chemical analysis at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media). Duplicate samples were taken and stored at approx. -20°C for further analysis if necessary.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Pre-study information indicated that the test material was pratically insoluble in water (<30 mg/L). Pre-study solubility work showed that the highest attainable test concentration that could be prepared (by visual inspection) ws 1.0 mg/L using a preliminary solution in tetrahydrofuran. at higher test concentrations precipitation of the test material was observed on addition of the test material solvent stock solution to water. Based on this information the test material was categorised as being a 'difficult substance' as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.

Saturated solution preparation:
An amount of test material (550 mg) was dispersed, in duplicate, in 11 litres of reconstituted water with the aid of propeller stirring at approx. 1500 rpm at a temperature of approx. 21°C for periods of either 24 or 48hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
- Centrifugation at 10000 g for 30 min
- Centrifugation at 40000 g for 30 min
- Filtration through a 0.2 µm Sartorius Sartopore filter (initial approx. 500 mL discarded in order to pre-condition the filter)
- Filtration through a 0.2 µm Sartorius Sartopore filter (initial approx. 1 L discarded in order to pre-condition the filter)

Solvent spike preparation:
An amount of test material (100 mg) was dissolved in tetrahydrofuran and the volume adjusted to 10 mL to give a 100 mg/10 mL solvent stock solution. An aliquot (1000 µL) of this solvent stock solution was dispersed in 10 litres of reconstituted water with the aid of magnetic stirring for approx. 10 minutes to give a 1.0 mg/L test concentration. Samples were taken for chemical analysis after the following pre-treatments:
- Untretaed
- Centrifugation at 10000 g for 30 min
- Centrifugation at 40000 g for 30 min
- Filtration through a 0.2 µm Gelman AcroCap filter (initial approx. 100 mL discarded in order to pre-condition the filter)
- Filtration through a 0.2 µm Gelman AcroCap filter (initial approx. 500 mL discarded in order to pre-condition the filter)

The remainder of the 1.0 mg/L test concentration was returned to the magnetic stirrer and stirred for a further 48 hours with samples being taken for analysis after both 24 and 48 hours stirring.

Range-finding test:
The results of the pre-study media preparation trial conducted indicated that a dissolved test material concentration of approx. 0.85 mg/L could be obtained using a solvent spike method of preparation with any undissolved test material removed by centrifugation at 40000 g for 30 minutes. The test concentration to be used in the difinitive test was determined by a preliminary range-finding test, where Daphnia magna were exposed to a series of nominal test concentrations of 0.0085, 0.085 and 0.85 mg/L. The test material was prepared using a preliminary solution in tetrahydrofuran. An amount of test material (100 mg) was dissolved in tetrahydrofuran and the volume adjusted to 10 mL to give a 100 mg/10 mL solvent stock solution. An aliquot (100 µL) of this solvent stock solution was dispersed in 1 litre of reconstituted water with the aid of magnetic stirring for approx. 10 minutes prior to removal of any undissolved test material by centrifugation at 40000 g for 30 minutes to give a 0.85 mg/L test concentration. A series of dilutions were prepared from the 0.85 mg/L test concentration to give further test concentrations of 0.085 and 0.0085 mg/L.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive test:
Based on the results of the range-fining test a "Limit test" was conducted at a nominal concentration of 0.85 mg/L to confirm that at the highest attainable test concentration no immobilisation or adverse reactions to exposure were observed.
For the purpose of the definitive test the test material was prepared using a preliminary solution in tetrahydrofuran. An amount of test material (100 mg) was dissolved in tetrahydrofuran and the volume adjusted to 10 mL to give a 100 mg/10 mL solvent stock solution. An aliquot (300 µL) of this solvent stock solution was dispersed in 3 litres of reconstituted water with the aid of magnetic stirring for approx. 30 minutes prior to removal of any undissolved test material by centrifugation at 40000 g for 30 minutes to give a nominal test concentration of 0.85 mg/L.
The stock solvent solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: in-house laboratory cultures
- Age at study initiation (mean and range, SD): 1st instar, <24 hours
- Method of breeding: Adult Daphnia maintained in polypropylene vessels containing approx. 2 litres of reconstituted water in a temperature controlled room at approx 20°C. The lightning 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. 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 culture and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity oe outcome of the study.
- Feeding during test: no

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

250 mg/L as CaCO3 (reconstituted water)

Test temperature:

20-21°C

pH:

7.8-8.0

Dissolved oxygen:

8.1-8.9 mg/L

Nominal and measured concentrations:

0.35 mg/L (time-weighted mean measured)
0.85 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: closed
- Material, size, headspace, fill volume: 250 mL, no headspace
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water: Stock solutions (11.76 g/L CaCl2.2H2O, 4.93 g/L MgSO4.7H2O, 2.59 g/L NaHCO3, 0.23 g/L KCl). Preparation: Aliquot (25 mL) of each of the stock solutions was added to each litre (final volume) of deionised water with a conductivity of <5 µS/cm. 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 approx. air-saturation value. The reconstituted water had an approx. theoretical total hardness of 250 mg/L as CaCO3.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light, 8 hours dark, 20 minutes transition periods of dusk and dawn


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.85, 0.085, and 0.0085 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.35 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

dissolved

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.85 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The test material was known to be unstable. Based on this the test was conducted using a semi static regime with chemical analysis of the test preparations being conducted at 0, 24 and 48 hours. Analysis of the freshly prepared test media at 0 and 24 hours showed measured test concentrations to range from 0.42 mg/L at 0 hours to 0.49 - 0.67 mg/L at 24 hours. A decline in measured concentration was observed in the old media at 24 and 48 hours with measured concentrations in the range of 0.23 to 0.25 mg/L at 24 hours and 0.22-0.24 mg/L at 48 hours. The decline in measured concentrations observed over the test period was in line with the preliminary stability analyses conducted. Given this decline in measured test concentrations it was considered justifiable to base the results on the time weighted mean measured test concentrations in order to give a "worst case" analysis of the data. The 48-hour EC50 based on the time-weighted mean measured test concentrations was greater than 0.35 mg/L and correspondingly the NOEC was 0.35 mg/L.

Table: Cumulative Immobilisation data in definitive test

Time-Weighted Mean Measured Test Concentration (mg/L)		Cumulative Immobilised Daphnia (Initial population: 5 per replicate)
		24 hours			48 hours
		No. per replicate	Total	%	No. per replicate	Total	%
Control	R1	0	0	0	0	1	5
	R2	0			0
	R3	0			0
	R4	0			1
Solvent Control	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0
0.35	R1	1	1	5	2	2	10
	R2	0			0
	R3	0			0
	R4	0			0

Validity criteria fulfilled:

yes

Conclusions:

48-hour EC50 > 0.85 mg/L, nominal

Executive summary:

A study according to OECD TG 202 was performed to assess the acute toxicity of PETMP to Daphnia magna.

Pre-study solubility work conducted indicated that the test material was insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. The highest dissolved test material that could be obtained (by visual inspection) was 1.0 mg/l using a preliminary solution in tetrahydrofuran. Based on this information the test material fell into the category of a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 23, 2000).

Pre-study media preparation trial indicated that the use of a solvent spike method of preparation followed by centrifugation to remove the undissolved test material was the most appropriate method of preparation for the test material giving a dissolved test material concentration of approximately 0.85 mg/l.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a time-weighted mean
measured test concentration of 0.35 mg/I for 48 hours at a temperature of approximately 20°C under semi static test conditions. Immobilisation 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 approximately 20°C under static test conditions. [mmobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

The test material was known to be unstable. Based on this the test was conducted using a semi static regime with chemical analysis of the test preparations
being conducted at 0, 24 and 48 hours.
Analysis of the freshly prepared test media at 0 and 24 hours showed measured test concentrations to range from 0.42 mg/l at 0 hours to 0.49 - 0.67 mg/i at 24 hours. A decline in measured concentration was observed in the old media at 24 and 48 hours with measured concentrations in the range of 0.23 to 0.25 mg/l at 24 hours and 0.22 - 0.24 mg/l at 48 hours.
The decline in measured concentrations observed over the test period was in line with the preliminary stability analyses conducted.
The 48-Hour EC50 based on the time weighted mean measured test concentrations was greater than 0.35 mg/l.
These values don't represent the real environmental conditions because the test substance is rapidly oxidized by the oxygen content in the aqueous phase.

Therefore, nominal values will be used for hazard assessment: the nominal EC50 was >0.85 mg/L.

Description of key information

EC50 (48h) > 0.85 mg/L (nominal) (Daphnia magna)

 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 0.85 mg/L

Additional information

A study according to OECD TG 202 was performed to assess the acute toxicity of PETMP to Daphnia magna.

Pre-study solubility work conducted indicated that the test material was insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. The highest dissolved test material that could be obtained (by visual inspection) was 1.0 mg/l using a preliminary solution in tetrahydrofuran. Based on this information the test material fell into the category of a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 23, 2000).

Pre-study media preparation trial indicated that the use of a solvent spike method of preparation followed by centrifugation to remove the undissolved test material was the most appropriate method of preparation for the test material giving a dissolved test material concentration of approximately 0.85 mg/l.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a time-weighted mean
measured test concentration of 0.35 mg/I for 48 hours at a temperature of approximately 20°C under semi static test conditions. Immobilisation 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 approximately 20°C under static test conditions. [mmobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

The test material was known to be unstable. Based on this the test was conducted using a semi static regime with chemical analysis of the test preparations
being conducted at 0, 24 and 48 hours.
Analysis of the freshly prepared test media at 0 and 24 hours showed measured test concentrations to range from 0.42 mg/l at 0 hours to 0.49 - 0.67 mg/i at 24 hours. A decline in measured concentration was observed in the old media at 24 and 48 hours with measured concentrations in the range of 0.23 to 0.25 mg/l at 24 hours and 0.22 - 0.24 mg/l at 48 hours.
The decline in measured concentrations observed over the test period was in line with the preliminary stability analyses conducted.
The 48-Hour EC50 based on the time weighted mean measured test concentrations was greater than 0.35 mg/l.
These values don't represent the real environmental conditions because the test substance is rapidly oxidized by the oxygen content in the aqueous phase.

Therefore, nominal values will be used for hazard assessment: the nominal EC50 was >0.85 mg/L.