Pyrolysis Reaction Product of Pinane

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

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 203 (Fish, Acute Toxicity Test)

Version / remarks:

adopted July 17, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

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 54.4 mg/L was analysed as highest concentration at the 4th renewal period as all fish died in the 100.0 mg/L concentration by that period. Three replicates were analysed from the test concentration and the control respectively at each analytical occasion.

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):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Akvárium magazin Kft. (Pasaréti Gyula) 1222 Budapest, Dévény u. 36.
- Length at study initiation: 1.55 - 2.35 cm
- Weight at study initiation: 0.09 - 0.11 g

ACCLIMATION
- Acclimation period: 12 days
- Acclimation conditions: same as test conditions
- Type and amount of food during acclimation: appropriate, commercial diet for fish
- Feeding frequency during acclimation: at least three times per week until one day before the test start
- Health during acclimation: no mortality observed

FEEDING DURING TEST
The fish will not be fed during the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

249 mg/L (as CaCO3).

Test temperature:

21.4 – 22.1°C

pH:

7.36 – 8.01

Dissolved oxygen:

65.1 – 89.2 %

Nominal and measured concentrations:

Nominal concentrations: 4.3, 9.4, 20.7, 45.5 and 100.0 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: 0.76, 1.67, 3.69, 8.11 and 17.8 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: aquarium
- Type: closed (sealed by glass plates)
- Material, headspace, fill volume: glass, no headspace, 2300mL
- Aeration: no
- Renewal rate of test solution: 24-h water renewal periods
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: reconstituted water (ISO medium, prepared according to recommendation of Annex 2 of the OECD 203 guideline

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light: 8 h dark

EFFECT PARAMETERS MEASURED
mortality at approximately 24, 48, 72 and 96 hours after start of the test

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
Range finding study
- Test concentrations: 0.1, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes, LC50 between 10 and 100 mg/L

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

10.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: recalculated based on the water solubility

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

3.69 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: recalculated based on the water solubility

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

8.11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: recalculated based on the water solubility

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

58.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: based on nominal loading rate

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

20.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: based on nominal loading rate

Duration:

96 h

Dose descriptor:

LOELR

Effect conc.:

45.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: based on nominal loading rate

Details on results:

No mortality was observed in the control during the test. The dissolved oxygen concentration in the test solutions did not fall below 60 % of air saturation value during the study. No significant change (more than one unit) in the pH value was observed during the test. All validity criteria were within acceptable limits and therefore the study was considered as valid.

Reported statistics and error estimates:

For determination of the LC50 value Probit analysis was performed using SPSS software. Other endpoints were determined directly from the raw data.

Sublethal observations / clinical signs:

Table 1: Cumulative mortality data

Concentration [mg/L loading rate]	Cumulative mortality (initial population = 7 fish / group)
	4h	24h	48h	53h	72h	73h	96h
Control	0	0	0	0	0	0	0
4.3	0	0	0	0	0	0	0
9.4	0	0	0	0	0	0	0
20.7	0	0	0	0	0	0	0
45.5	0	0	0	0	0	0	1
100.0	0	2	5	7	7	7	7

 

Table 2: Sub-lethal effects observed during the study

Concentration [mg/L loading rate]	Symptoms	Numbers of fish / Observation time
		4h	24h	48h	53h*	72h	73h^#	96h
Control	–	–	–	–		–		–
	Cumulative mortality	0	0	0	0	0	0	0
4.3	–	–	–	–		–		–
	Cumulative mortality	0	0	0	0	0	0	0
9.4	–	–	–	–		–		–
	Cumulative mortality	0	0	0	0	0	0	0
20.7	–	–	–	–		–		–
	Cumulative mortality	0	0	0	0	0	0	0
45.5	Slightly reduced activity (observed only after water renewal, in fresh test solution)	7	7	7		7	0	0
	Slightly reduced activity (observed in old test solution, before water renewal as well)	0	0	0		0	0	0
	Reduced activity	0	0	4		5	3	6
	Lying at the bottom of aquarium	0	0	0		0	4	0
	Cumulative mortality	0	0	0	0	0	0	1
100.0	Severely reduced activity; off-balance; swimming only periodically; lying at the bottom of aquarium;	7	4	2
	Lying at the bottom of aquarium; gill movements only	0	1	0
	Cumulative mortality	0	2	5	7	7	7	7

–   no sign of symptoms

*   53-h observation period was included only in concentration of 100 mg/L (loading rate)

^#    73-h observation period was included only in concentration of 45.5 mg/L (loading rate)

Table 3: 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		3^rdrenewal period		4^threnewal period
	start	end	start	end	start	end	start	end
100.0	4.49	3.32	4.03	4.19	3.50*	3.16	–	–
45.5	–	–	–	–			1.44*	0.57*

* 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 mortality of Danio rerio after 96 hours with the following effect values: The EC50 value was 10.4 mg/L, recalculated based on the water solubility of the test item.

Executive summary:

The purpose of this study according to OECD guideline 203 was to evaluate the influence of the test item on zebrafish (Danio rerio). Young fish were exposed in an acute toxicity test to aqueous test media containing the test item for 96 hours at a range of concentration in a semi-static test system (daily renewal periods). The test method of application and the test species Zebrafish (Danio rerio) are recommended by the test guidelines. Based on the results of the preliminary experiment, nominal concentrations of 4.3, 9.4, 20.7, 45.5 and 100.0 mg/L (loading rate) were investigated in the main study. Biological results are based on the nominal loading rates. Seven fish were exposed to each concentration and a control for 96 hours. The fish were observed at least on each day of the test for signs of intoxication and mortality. Body weights of fish were determined prior to test initiation; body length measurement was performed at the end of the test (or when any mortality occurred). Measurements of environmental conditions were performed daily (at the start and end of each water renewal period). Sealed glass aquariums filled up fully with the test solution (thus allowing no headspace) were used as test vessels. The volume of the test liquid in the vessels was approx. 2300 mL. 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 LL50, NOELR and LOELR for 96-h test period. For determination of the LL50 value 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 can be considered as valid. The measured mean total organic carbon (TOC) concentration was 4.49 mg/L at the start and 0.57 mg/L (below the quantification limit) at the end of the test. In this 96-hour semi-static acute toxicity test on Zebrafish (Danio rerio) the test item had significant toxic effects on fish. The 96-h LL50 value was determined to be 58.4 mg/L (loading rate). The NOELR was determined to be 20.7 mg/L (loading rate) and the LOELR was determined to be 45.5 mg/L (loading rate). All results were recalculated based on the water solubility of the test item, the LC50 was determined to be 10.4 mg/L, the NOEC 3.69 mg/L and the LOEC 8.11 mg/L.

Description of key information

In a semi-static short term study the test item was found to affect the mortality of Danio rerio after 96 hours with the following effect values: The EC50 value was 10.4 mg/L, recalculated based on the water solubility of the test item.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

10.4 mg/L

Additional information

The purpose of this study according to OECD guideline 203 was to evaluate the influence of the test item on zebrafish (Danio rerio). Young fish were exposed in an acute toxicity test to aqueous test media containing the test item for 96 hours at a range of concentration in a semi-static test system (daily renewal periods). The test method of application and the test species Zebrafish (Danio rerio) are recommended by the test guidelines. Based on the results of the preliminary experiment, nominal concentrations of 4.3, 9.4, 20.7, 45.5 and 100.0 mg/L (loading rate) were investigated in the main study. Biological results are based on the nominal loading rates. Seven fish were exposed to each concentration and a control for 96 hours. The fish were observed at least on each day of the test for signs of intoxication and mortality. Body weights of fish were determined prior to test initiation; body length measurement was performed at the end of the test (or when any mortality occurred). Measurements of environmental conditions were performed daily (at the start and end of each water renewal period). Sealed glass aquariums filled up fully with the test solution (thus allowing no headspace) were used as test vessels. The volume of the test liquid in the vessels was approx. 2300 mL. 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 LL50, NOELR and LOELR for 96-h test period. For determination of the LL50 value 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 can be considered as valid. The measured mean total organic carbon (TOC) concentration was 4.49 mg/L at the start and 0.57 mg/L (below the quantification limit) at the end of the test. In this 96-hour semi-static acute toxicity test on Zebrafish (Danio rerio) the test item had significant toxic effects on fish. The 96-h LL50 value was determined to be 58.4 mg/L (loading rate). The NOELR was determined to be 20.7 mg/L (loading rate) and the LOELR was determined to be 45.5 mg/L (loading rate). All results were recalculated based on the water solubility of the test item, the LC50 was determined to be 10.4 mg/L, the NOEC 3.69 mg/L and the LOEC 8.11 mg/L.