Reaction mass of m-terphenyl and o-terphenyl

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:

The experiment was performed in following time period: 2017-12-12 to 2018-03-17.

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)

Version / remarks:

Version of April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Version of 2008, as published in Commission Regulation (EC) No 440/2008.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The department of health of the government of the United Kingdom (2017-11-28)

Analytical monitoring:

yes

Remarks:

The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0, 24 and 48 hours.

Details on sampling:

- 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% v/v saturated solution. Based on the results of the range-finding test an initial experiment was conducted at a concentration of 100% v/v saturated solution to confirm that at highest attainable test concentration no immobilization or adverse reactions to exposure were observed. Definitive study: Chemical analysis of the freshly prepared 100% v/v saturated solution test preparations at 0 and 24 hours showed measured test concentrations to range from 0.013 to 0.019 mg/L. Analysis of the corresponding old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to between 0.0040 and 0.0076 mg/L (32% to 40% of the fresh measured test concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data. The geometric mean measured test concentration was determined to be 0.0096 mg/L.
- Sampling method: Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.
- Sample storage conditions before analysis: All samples (for verification of test concentrations) were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Based on this information the test item was categorized 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 item under test conditions.
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.062 mg/L could be obtained using a saturated solution method of preparation. The test concentration to be used in the definitive test was 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% v/v saturated solution.

- Controls: The control group was maintained under identical conditions but not exposed to the test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): No vehicle used.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No evidenece for such effects.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM:
- Common name: Daphnia magna
- Strain/clone: Not reported.
- Justification for species other than prescribed by test guideline: Not applicable.
- Source: The daphnids were derived from in-house laboratory cultures.
- Age of parental stock (mean and range, SD): The test was carried out using first instar Daphnia magna. 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: No feeding during study.

ACCLIMATION:
- Acclimation period: No acclimation with the test daphnids performed. The test was carried out using first instar Daphnia magna, less than 24 hours old. The adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium.
- Type and amount of food: Each culture of the adult daphnids was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension.
- Feeding frequency: Daily.

QUARANTINE (wild caught): Not applicable.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures. Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22°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.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

Test media renewal after 24 hours, i.e. semi-static conditions

Post exposure observation period:

No additional post exposure period implemented in test design.

Hardness:

Not reported.

Test temperature:

The test vessels were maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1°C. The water temperature was recorded daily throughout the test.

pH:

pH values were between 8.0 and 8.1. The pH values were recorded daily throughout the test.

Dissolved oxygen:

8.7 to 9.1 mg O2/L. The dissolved oxygen concentrations were recorded daily throughout the test.

Salinity:

Not relevant for freshwater study.

Nominal and measured concentrations:

The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0, 24 and 48 hours. Chemical analysis of the freshly prepared 100% v/v saturated solution test preparations at 0 and 24 hours showed measured test concentrations to range from 0.013 to 0.019 mg/L. Analysis of the corresponding old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to between 0.0040 and 0.0076 mg/L (32% to 40% of the fresh measured test concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data.

Details on test conditions:

TEST SYSTEM
- Test vessel: As in the range-finding test 150 mL glass beakers containing approximately 100 mL of test preparation were used.
- Type (delete if not applicable): Closed; the test vessels then covered to reduce evaporation
- Material, size, headspace, fill volume: Glass, 100 mL fill volume
- Aeration: Not performed.
- Type of flow-through (e.g. peristaltic or proportional diluter): No flow-through conditions. Semi-static conditions implemented (change of medium after 24 h).
- Renewal rate of test solution (frequency/flow rate): Change of medium after 24 h. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.
- No. of organisms per vessel: At the start of the test five daphnids were placed in each test and control vessel at random, in the test preparations.
- No. of vessels per concentration (replicates): Four replicate test vessels were prepared.
- No. of vessels per control (replicates): Four replicate control vessels were prepared.
- No. of vessels per vehicle control (replicates): Not applicable.
- Biomass loading rate: Not applicable.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (Elendt M7 medium) used for the range-finding test, initial experiment and definitive test. The pH of the prepared media was 7.9 ±0.3.
- Total organic carbon: Information not available.
- Particulate matter: Information not available.
- Metals: Information not available, defined by composition of Elendt M7 medium, as used in this study.
- Pesticides: Information not available.
- Chlorine: Information not available, defined by composition of Elendt M7 medium, as used in this study.
- Alkalinity: The pH of the prepared media was 7.9 ± 0.3, further information not available.
- Ca/Mg ratio: Information not available, defined by composition of Elendt M7 medium, as used in this study.
- Conductivity: Information not available.
- Salinity: Information not available.
- Culture medium different from test medium: No.
- Intervals of water quality measurement: The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. Samples were taken for quantitative analysis of test compound from the freshly prepared control and 100% v/v saturated solution test preparation at 0 and 24 hours and from the corresponding old or expired media (replicates pooled) at 24 and 48 hours.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: Photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Light intensity: Between 200 and 1200 Lux.

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 within 15 seconds after gentle agitation.

VEHICLE CONTROL PERFORMED: Not applicable, no vehicle used.

RANGE-FINDING STUDY
- Test concentrations: The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.062 mg/L could be obtained using a saturated solution method of preparation. The test concentration to be used in the definitive test was 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% v/v saturated solution.
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test an initial experiment was conducted at a concentration of 100% v/v saturated solution to confirm that at highest attainable test concentration no immobilization or adverse reactions to exposure were observed. Significant immobilization was observed to have occurred after 48 hours exposure and hence the test was repeated.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate was used as reference item.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.01 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

Remarks:

No immobilization was observed throughout the test.

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.01 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

Remarks:

This study showed that there were no toxic effects at saturation

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 0.01 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: No immobilization was observed throughout the test.

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

0.01 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: No immobilization was observed throughout the test.

Details on results:

- Behavioural abnormalities: In the range-finding study, trapping of the daphnids at the surface was observed in the 10% v/v saturated solution replicate R2.
- Observations on body length and weight: No.
- Other biological observations: None.
- Mortality of control: Only 1 immobilized daphnid observed (in container R4).
- Other adverse effects control: No.
- Abnormal responses: No abnormal responses.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No. At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.
- Effect concentrations exceeding solubility of substance in test medium: Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.
A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.062 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this test item under test conditions.

Results with reference substance (positive control):

- Results with reference substance valid? Yes. A positive control (Envigo study number LK67NP) used potassium dichromate as the reference item. The positive control was conducted between 23 January 2018 and 25 January 2018.
- Relevant effect levels: Results for 24 h exposure period: EC50 = 0.79 mg/L, NOEC = 0.56 mg/L, LOEC = 1.0 mg/L.
Results for 48 h exposure period: EC50 = 0.75 mg/L, NOEC = 0.56 mg/L, LOEC = 1.0 mg/L.
- Limit test: No
- Dose-response test: Yes.

Reported statistics and error estimates:

An estimate of the EC50 values was given by inspection of the immobilization data. The geometric mean measured concentrations for each period of media renewal (0-24 and 24-48 hours) were calculated and the arithmetic mean of the two values determined. The computerized systems used in this project were Shimadzu GCMS solution 2.50 SU1 and the Delta building management system.

Table 1: Cumulative Immobilization Data and Observations in the Range-finding Test

	Observations (Initial Population: 5 Per Replicate)
	24 h				48 h
Concen- tration*	Cumulative Immobilized Daphnia		Observations		Cumulative Immobilized Daphnia		Observations
	R1	R2	R1	R2	R1	R2	R1	R2
Control	0	0	5 N	5 N	0	0	5 N	5 N
0.10	0	0	5 N	5 N	0	0	5 N	5 N
1.0	0	0	5 N	5 N	0	0	5 N	5 N
10	0	0	5 N	2 N, 3 T	0	0	5 N	4 N, 1 T
100	0	0	5 N	5 N	0	0	5 N	5 N

*: Nominal concentration (% v/v Saturated Solution)

R = Replicate; N = Normal; T = Trapped at surface

Table 2 Cumulative Immobilization Data and Observations in the Definitive Test

Concen- tration*	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
	24 hours
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.0096	0	0	0	0	0	0	5 N	5 N	5 N	5 N
	48 hours
Control	0	0	0	1	1	5	5 N	5 N	5 N	4 N
0.0096	0	1	0	0	1	5	5 N	4 N	5 N	5 N

*: Geometric mean measured test concentration (mg/L)

R = Replicate; N = Normal

Validity criteria fulfilled:

yes

Conclusions:

Acute toxicity of the compound reaction mass of m-terphenyl and o-terphenyl was investigated under semistatic conditions. The test was performed according to OECD Guideline 202 (2004) and EU Method C.2 (2008). Exposure of Daphnia magna to the test item gave EC50 (48 h) values based on the geometric mean measured test concentration of greater than 0.0096 mg/L. The NOEC (48 h) was 0.0096 mg/L. No immobilization was observed throughout the test. This study showed that there were no toxic effects at saturation.

Executive summary:

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. It was conducted following GLP.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.062 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this test item under test conditions.

Following a preliminary range-finding test and initial experiment, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a concentration of 100% v/v saturated solution for 48 hours at a temperature of 19 °C to 22 °C under semi-static test conditions. The test item solution was prepared by stirring an excess (50 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution of the test item. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media. The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0, 24 and 48 hours.

Results: Chemical analysis of the freshly prepared 100% v/v saturated solution test preparations at 0 and 24 hours showed measured test concentrations to range from 0.013 to 0.019 mg/L. Analysis of the corresponding old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to between 0.0040 and 0.0076 mg/L (32% to 40% of the fresh measured test concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data. The geometric mean measured test concentration was determined to be 0.0096 mg/L.

Exposure of Daphnia magna to the test item gave EC50 values based on the geometric mean measured test concentration of greater than 0.0096 mg/L. The No Observed Effect Concentration (NOEC) was 0.0096 mg/L. No immobilization was observed throughout the test. This study showed that there were no toxic effects at saturation.

Description of key information

OECD 202, GLP, D. magna, semi-static. EC50 (48 h) > 0.0096 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.01 mg/L

Additional information

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.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.062 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this test item under test conditions.

Following a preliminary range-finding test and initial experiment, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a concentration of 100% v/v saturated solution for 48 hours at a temperature of 19 °C to 22 °C under semi-static test conditions. The test item solution was prepared by stirring an excess (50 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution of the test item. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media. The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0, 24 and 48 hours.

Results: Chemical analysis of the freshly prepared 100% v/v saturated solution test preparations at 0 and 24 hours showed measured test concentrations to range from 0.013 to 0.019 mg/L. Analysis of the corresponding old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to between 0.0040 and 0.0076 mg/L (32% to 40% of the fresh measured test concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data. The geometric mean measured test concentration was determined to be 0.0096 mg/L.

Exposure of Daphnia magna to the test item gave EC50 values based on the geometric mean measured test concentration of greater than 0.0096 mg/L. The No Observed Effect Concentration (NOEC) was 0.0096 mg/L. No immobilization was observed throughout the test. This study showed that there were no toxic effects at saturation.