Pyrolysis Reaction Product of Pinane

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:

May 2019

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:

adopted 13 April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Version / remarks:

October 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Amount of total organic carbon (TOC) was determined in the highest test concentration and the control at the start and at the end of each water renewal period. The test concentration of 45.5 mg/L was analysed as highest concentration at the 2nd renewal period as all Daphnia were observed as immobile in the 100.0 mg/L concentration at the 24-h observation period. Three replicates were analysed from the test concentration and the control respectively at each analytical occasion. Measured control values were used as blank for calculation of TOC concentration of test item treated samples.

Vehicle:

no

Details on test solutions:

The test item is a poorly water soluble UVCB material therefore a water-accommodated fraction (WAF) was prepared as follows: a supersaturated solution (100 mg/L nominal loading) was prepared by dispersing/dissolving the test item amount into the test medium (ISO medium) in a sealed Erlenmeyer flask filled up fully, thus allowing no headspace. This solution was stirred for 24 hours using magnetic stirrer and then settle for approx. one hour to allow phase separation. The saturated solution was removed from the approximate middle of flask. The test solutions of subsequent lower test concentrations was prepared by appropriate dilution of this stock WAF.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water fleas
- Clone: Daphnia magna (Straus)
- Age at study initiation: Less than 24 hours old
- Source: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100 Gödöllő, Kotlán S. u. 3. Hungary
- Feeding during test : no

ACCLIMATION
- Acclimation period: no

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

249 mg/L (as CaCO3)

Test temperature:

20.9 – 21.3°C

pH:

7.59 – 8.24

Dissolved oxygen:

7.88 – 7.83 mg/L

Nominal and measured concentrations:

Nominal concentrations: 1.9, 4.3, 9.4, 20.7, 45.5 and 100 mg/L (loading rate), no measured concentrations
The nominal concentrations were recalculated based on the water solubility of 17.817 mg/L (see IUCLID section 4.8). The calculated concentrations were: 0.34, 0.77, 1.67, 3.69, 8.11 and 17.8 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Type: closed (sealed by parafilm and aluminium foil)
- Material, headspace, fill volume: glass, no headspace, 50 mL
- Volume of solution: 50 mL
- Aeration: no
- Renewal rate of test solution: 24-h renewal periods
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: ISO Medium, according to OECD 202

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light (artificial illumination) and 8 hours darkness
- Light intensity: 723 lux

EFFECT PARAMETERS MEASURED
immobility determined by visual observation after 24 and 48 hours,
Temperature, pH, oxygen concentrations measured at start and end of test

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1, 10, 100 mg/L nominal loading
- Results used to determine the conditions for the definitive study: yes, EC50 between 10 and 100 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: recalculated based on the water solubility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

2.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: recalculated based on the water solubility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.67 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: recalculated based on the water solubility

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

24.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: based on nominal loading rate

Duration:

48 h

Dose descriptor:

EL10

Effect conc.:

16.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: based on nominal loading rate

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

9.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: based on nominal loading rate

Details on results:

The immobilisation rate of the introduced Daphnia did not exceed 10 %. Immobility was not observed in the untreated control and no Daphnia were trapped at the water surface or showed any sign of stress. The dissolved oxygen concentration at the end of the test was more than 3 mg/L in control and test vessels. All validity criteria were within acceptable limits and therefore the study was considered as valid.

Results with reference substance (positive control):

The date of the last study (Study Number: 392-202-4466) with reference item Potassium dichromate was: 12 – 13 March 2019. The 24h EC50: 1.58 mg/L, (95 % confidence limits: 1.34 – 1.85 mg/L)

Reported statistics and error estimates:

The 48-h ECx values were calculated by Probit analysis using SPSS software. The NOEC, LOEC values were determined directly from the raw data.

Table 1: Immobilisation of the test animals

Concentration [mg/L loading rate]	No. of Daphnia tested	No. of immobilised Daphnia after		% of immobilised Daphnia after
		24 h	48 h	24 h	48 h
Control	20	0	0	0	0
1.9	20	0	0	0	0
4.3	20	0		0	0
9.4	20	0	0	0	0
20.7	20	0	5	0	25
45.5	20	0	20	0	100
100.0	20	20	20	100	100

Table 2: Total organic carbon (TOC) concentrations measured during the test

Nominal concentration [mg/L loading rate]	Measured TOCconcentration [mg/L]
	1^strenewal period		2^ndrenewal period
	start	end	start	end
100.0	3.55*	3.26	–	–
45.5	–	–	1.96*	2.44*

*these values were below the quantification limit (< BQL) based on the actual daily calibration curves

Validity criteria fulfilled:

yes

Conclusions:

In a semi-static short term study the test item was found to affect the mobility of Daphnia magna after 48 hours with the following effect values: The EC50 and EC10-values were 4.4 mg/L and 2.9 mg/L, recalculated based on the water solubility of the test item.

Executive summary:

The purpose of this study according to OECD guideline 202 was to evaluate the influence of the test item on the mobility respectively survival of Daphnia magna. Young Daphnia were exposed in an immobilization test to aqueous test media containing the test item for 48 hours at a range of concentration under semi-static conditions. Based on the results of the preliminary experiment, nominal concentrations of 1.9, 4.3, 9.4, 20.7, 45.5 and 100 mg/L (loading rate) were investigated in the main study. Concurrent control ran. Biological results are based on the nominal loading rates.The test item and the control were tested using 20 Daphnia, divided into four groups of five animals (glass beaker; approx. 50 mL test medium) each were exposed to the test item concentrations or run as control for 48 hours in a semi-static test (24-h renewal periods). The test vessels were sealed and filled up fully with the test solution (thus allowing no headspace). The immobility or mortality of the Daphnia was determined by visual observation after 24 and 48 hours of exposure. Amount of total organic carbon (TOC) was determined in the highest test concentration and the control at the start and at the end of each water renewal period immediately after sampling. The endpoints used in this study are EL10, EL50 and NOELR for 48h test period. For determination of the ELx values Probit analysis was performed using SPSS software. Other endpoints were determined directly from the raw data. All validity criteria were met and therefore the study is considered as valid. Total organic carbon (TOC) concentrations measured during the test are mostly below quantification limit, only after 24h 3.26 mg/L were above the limit. The following results for the immobilization of daphnia were determined: EL50 = 24.9 mg/L, EL10 = 16.7 mg/L, NOELR = 1.67 mg/L. The results were recalculated based on the water solubility of the test item: EC50 = 4.4 mg/L, EC10 = 2.9 mg/L, NOEC = 1.67 mg/L.

Description of key information

In a semi-static short term study the test item was found to affect the mobility of Daphnia magna after 48 hours with the following effect values: The EC50 and EC10-values were 4.4 mg/L and 2.9 mg/L, recalculated based on the water solubility of the test item.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

4.4 mg/L

Additional information

The purpose of this study according to OECD guideline 202 was to evaluate the influence of the test item on the mobility respectively survival of Daphnia magna. Young Daphnia were exposed in an immobilization test to aqueous test media containing the test item for 48 hours at a range of concentration under semi-static conditions. Based on the results of the preliminary experiment, nominal concentrations of 1.9, 4.3, 9.4, 20.7, 45.5 and 100 mg/L (loading rate) were investigated in the main study. Concurrent control ran. Biological results are based on the nominal loading rates.The test item and the control were tested using 20 Daphnia, divided into four groups of five animals (glass beaker; approx. 50 mL test medium) each were exposed to the test item concentrations or run as control for 48 hours in a semi-static test (24-h renewal periods). The test vessels were sealed and filled up fully with the test solution (thus allowing no headspace). The immobility or mortality of the Daphnia was determined by visual observation after 24 and 48 hours of exposure. Amount of total organic carbon (TOC) was determined in the highest test concentration and the control at the start and at the end of each water renewal period immediately after sampling. The endpoints used in this study are EL10, EL50 and NOELR for 48h test period. For determination of the ELx values Probit analysis was performed using SPSS software. Other endpoints were determined directly from the raw data. All validity criteria were met and therefore the study is considered as valid. Total organic carbon (TOC) concentrations measured during the test are mostly below quantification limit, only after 24h 3.26 mg/L were above the limit. The following results for the immobilization of daphnia were determined: EL50 = 24.9 mg/L, EL10 = 16.7 mg/L, NOELR = 1.67 mg/L. The results were recalculated based on the water solubility of the test item:EC50 = 4.4 mg/L, EC10 = 2.9 mg/L, NOEC = 1.67 mg/L.