Pyrolysis Reaction Product of Pinane

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

adopted 23rd March, 2006, corrected 28 July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

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 the test. 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 be prepared as follows: a supersaturated solution (100 mg/L nominal loading) was prepared by dispersing/dissolving the test item amount into the test medium (OECD 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 settled 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):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: SAG, Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Age of inoculum: 2-4 days
- Method of cultivation: on agar

ACCLIMATION
- Acclimation period: no
- Culturing media and conditions: same as test conditions
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.3 – 22.6 °C

pH:

7.46 – 10.60

Nominal and measured concentrations:

Nominal concentration: 0.3, 1.0, 3.1, 9.8, 31.3 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: 0.05, 0.18, 0.55, 1.75, 5.58 and 17.8 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed (sealed by parafilm and aluminium foil)
- Material, headspace, fill volume: glass, no headspace, 114 mL
- Aeration: no
- Initial cells density: 10^4 cells/mL
- Control end cells density: 27.33 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD medium

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: OECD Medium, according to OECD 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: no
- Photoperiod: continuously
- Light intensity and quality: 6171 lux, fluorescent lamps (with a spectral range of 400 - 700 nm)

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: with counting chamber after 24, 48 and 72 hours
- Other: Temperature every 24h, pH at start and end of the test, light intensity at start of test

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
Range finding study:
- Test concentrations: 0.1, 1, 10, 100 mg/L nominal loading rate
- Results used to determine the conditions for the definitive study: yes, the EC50 was between 1 and 100 mg/L for Inhibition of growth and yield.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: recalculated based on the water solubility

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: recalculated based on the water solubility

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: recalculated based on the water solubility

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: recalculated based on the water solubility

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: recalculated based on the water solubility

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: recalculated based on the water solubility

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: recalculated based on the water solubility

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: recalculated based on the water solubility

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

48.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on nominal loading rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

18.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: based on nominal loading rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

4.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on nominal loading rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: based on nominal loading rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

9.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on nominal loading rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: based on nominal loading rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

31.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on nominal loading rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: based on nominal loading rate

Details on results:

The pH of the control cultures varied by more than 1.5 units during the test (mean difference: 3.1 units). However, this higher pH drift was considered to be due to the closed system used for the test by ensuring less CO2 mass transfer rate from the surrounding air. The closed system (i.e. sealed test vessels) was used for maintaining the test item concentrations as constant as possible and therefore the drift of pH can be considered to be acceptable and have not any effect on the output of the study.
Cell density in the control cultures increased by a factor of more than 16 (by a factor of 27.33) within 72 hours. This corresponds to a specific growth rate of 1.10 day-1. The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures did not exceed 35 %. CV for section-by-section growth rate day 0-1: 31.15 %; CV for section-by-section growth rate day 1-2: 16.40 %; CV for section-by-section growth rate day 2-3: 15.83 %. The mean coefficient of variation for section-by-section specific growth rates: 21.13 %. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 % in test. CV for average specific growth rate day 0-3: 3.58 %. All validity criteria were met, therefore, the study is considered as valid.

Results with reference substance (positive control):

The date of the last study (Study Number: 392-201-4465) with the reference item Potassium dichromate was: 11 – 14 March 2019. The following results were obtained: The 72h ErC50: 0.83 mg/L (95 % confidence limits: could not be calculated). The 72h EyC50: 0.57 mg/L (95 % confidence limits: 0.45 – 0.72 mg/L).

Reported statistics and error estimates:

Mean values and standard deviations of cell concentrations were calculated for each treatment at the start, after 24 h and 48 h, and at the end of the test (72 hours after the start of the test) using Excel for Windows software. Percentage inhibition of growth rate (µ) and yield (y) were calculated using Excel for Windows software. The ELx values and their confidence limits for growth rate and yield were calculated using Probit analysis by SPSS software. For the determination of the LOELR and NOELR, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values using analysis of variance (ANOVA) and Dunnett’s test (Dunnett T3; α = 0.05) by SPSS software.

Table 1: Growth Rates (µ), Yield (y) and percentage inhibition of µ during the test period

Concentration [mg/L loading rate]	Growth Rates (µ) and % Inhibition of µ						Yield y and % Inhibition of y
	0–24 h		0–48 h		0–72 h		0–72 h
	µ	%	µ	%	µ	%	y	%
Control	0.0393	–	0.0583	–	0.0459	–	26.33	–
0.3	0.0401	-2.1	0.0577	1.2	0.0445	2.9	23.67	10.1
1.0	0.0345	12.2	0.0601	-3.1	0.0435	5.1	22.00	16.5
3.1	0.0193	51.0	0.0615	-5.5	0.0416	9.4	19.00	27.8
9.8	0.0000	100.0	0.0573	1.8	0.0416	9.4	19.00	27.8
31.3	0.0193	51.0	0.0541	7.2	0.0409	10.9	18.00	31.6
100.0	0.0000	100.0	0.0000	100.0	0.0032	93.0	0.33	98.7

 

Table 2: Morphological changes of algal cells

Concentration [mg/L loading rate]	Symptoms
	24 h	48 h	72 h
Control	normal cells	normal cells	normal cells
0.3	normal cells	normal cells	normal cells
1.0	normal cells	normal cells	normal cells
3.1	normal cells	normal cells	normal cells
9.8	normal cells	normal cells	normal cells
31.3	normal cells	normal cells	normal cells
	swollen cells	swollen cells	swollen cells
100.0	normal cells	normal cells	normal cells
	swollen cells	swollen cells	swollen cells

Validity criteria fulfilled:

yes

Conclusions:

In a static short term study the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values: The EC50 and EC10-values for inhibition of growth rate were 8.7 mg/L and 0.8 mg/L respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 1.7 mg/L and 0.05 mg/L, respectively. All values were recalculated based on the water solubility of the test item.

Executive summary:

The purpose of this study according to OECD guideline 201 was to determine the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum). The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test method of application and the test species Raphidocelis subcapitata are recommended by the test guidelines. Based on the results of the preliminary experiment, nominal concentrations of 0.3, 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L (loading rate) were investigated in the main study. Biological results are based on the nominal loading rates, recalculated with the water solubility. The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 10^4 cells/mL at the start of the test in all of the test cultures. Sealed glass Erlenmeyer flasks with a total capacity of approx. 100-120 mL filled up fully, thus allowing no headspace were used as test vessels. The volume of the test liquid in the vessels was 114 mL. The alga cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals. Amount of total organic carbon (TOC) was determined in the highest test concentration and the control at the start and at the end of the test immediately after sampling. The endpoints used in this study are EL10, EL50, NOELR and LOELR for both response variables (i.e. average specific growth rate and yield). For the determination of the LOELR and NOELR, ANOVA and Dunnett’s test was used and for the determination of the ELx values Probit analysis was used (by SPSS software) if necessary. All validity criteria were met and, therefore, the study is considered as valid. The measured mean total organic carbon (TOC) concentrations of the highest test concentration (100.0 mg/L loading rate) was 4.07 mg/L at the start and 2.44 mg/L (below the quantification limit) at the end of the test. The following results for the inhibition of algae were determined: EL50 = 48.8 mg/L, EL10 = 4.4 mg/L, NOELR = 9.8 mg/L, LOELR = 31.1 mg/L for growth rate. The results for yield were: EL50 = 18.8 mg/L, EL10 = 0.6 mg/L, NOELR = 0.3 mg/L, LOELR = 1.0 mg/L. The results were recalculated based on the water solubility to be: EC50 = 8.7 mg/L, EC10 = 0.8 mg/L, NOEC = 1.7 mg/L, LOEC = 5.6 mg/L for growth rate. The results for yield were: EC50 = 3.4 mg/L, EC10 = 0.1 mg/L, NOEC = 0.05 mg/L, LOEC = 0.2 mg/L.

Description of key information

In a static short term study the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values: The EC50 and EC10-values for inhibition of growth rate were 8.7 mg/L and 0.8 mg/L respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 1.7 mg/L and 0.05 mg/L, respectively. All values were recalculated based on the water solubility of the test item.

Key value for chemical safety assessment

EC50 for freshwater algae:

8.7 mg/L

EC10 or NOEC for freshwater algae:

0.8 mg/L

Additional information

The purpose of this study according to OECD guideline 201 was to determine the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum). The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test method of application and the test species Raphidocelis subcapitata are recommended by the test guidelines. Based on the results of the preliminary experiment, nominal concentrations of 0.3, 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L (loading rate) were investigated in the main study. Biological results are based on the nominal loading rates, recalculated with the water solubility. The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 10^4 cells/mL at the start of the test in all of the test cultures. Sealed glass Erlenmeyer flasks with a total capacity of approx. 100-120 mL filled up fully, thus allowing no headspace were used as test vessels. The volume of the test liquid in the vessels was 114 mL. The alga cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals. Amount of total organic carbon (TOC) was determined in the highest test concentration and the control at the start and at the end of the test immediately after sampling. The endpoints used in this study are EL10, EL50, NOELR and LOELR for both response variables (i.e. average specific growth rate and yield). For the determination of the LOELR and NOELR, ANOVA and Dunnett’s test was used and for the determination of the ELx values Probit analysis was used (by SPSS software) if necessary. All validity criteria were met and, therefore, the study is considered as valid. The measured mean total organic carbon (TOC) concentrations of the highest test concentration (100.0 mg/L loading rate) was 4.07 mg/L at the start and 2.44 mg/L (below the quantification limit) at the end of the test. The following results for the inhibition of algae were determined: EL50 = 48.8 mg/L, EL10 = 4.4 mg/L, NOELR = 9.8 mg/L, LOELR = 31.1 mg/L for growth rate. The results for yield were: EL50 = 18.8 mg/L, EL10 = 0.6 mg/L, NOELR = 0.3 mg/L, LOELR = 1.0 mg/L. The results wererecalculated based on the water solubility to be:EC50 = 8.7 mg/L, EC10 = 0.8 mg/L, NOEC = 1.7 mg/L, LOEC = 5.6 mg/L for growth rate. The results for yield were: EC50 = 3.4 mg/L, EC10 = 0.1 mg/L, NOEC = 0.05 mg/L, LOEC = 0.2 mg/L.