(1R,5R)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 April 2019 – 18 April 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well conducted and well described study in accordance with GLP and OECD Guideline 201. However, pH level in the control and treatment groups varied more than 1.5 units at the end of the test and chemical analyses revealed severe test item losses during the test.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

yes

Remarks:

pH level in the control varied more than 1.5 units at the end of the test. However, the algal growth was sufficient and the validity criteria of the increase biomass during 72 hours was upper than 16. This deviation has no incidence on study results.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 15%, 25%, 40%, 62.5% and 100% (nominal concentrations expressed in % v/v saturated solution)
- Sampling method: At initial time (T0h), all the solutions were analysed taken directly from the starting solutions without algae. For other times, samples were taken from one flask . Approximately 1 mL was taken for each sample, then put in a vial and analysed by HPLC-UV.
- Sample storage conditions before analysis: All the solutions were kept in refrigerator or freeze.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was found poorly soluble and volatile, then a special solubilisation protocol in the test medium (OECD medium) was carried out before the experimentation, according to the OECD nº23 guidance document on aquatic toxicity testing of difficult substances and mixtures.
The mixing protocol consisted in three independent stock solutions at 100 mg/L (nominal concentration) which were stirred slowly 24 hours at 20 ± 2°C, at darkness and without headspace. After this step, a settling period of 30 minutes was performed and then sampled at the bottom of the flask after removing the first 100 mL. The three solutions were pooled to obtain a stock solution with sufficient volume to prepare the serial dilution. This stock solution was considered as 100% of saturation concentration. A range by serial dilution in a geometric series was prepared from this stock solution: 100% - 62.5% - 40% - 25% and 15% (expressed as % v/v saturated solution).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga Pseudokirchneriella subcapitata
- Strain: chlorococcal order strain number CCAP 278.4
- Source (laboratory, culture collection): Not reported.
- Method of cultivation: Not reported.

ACCLIMATION
- Acclimation period: Not reported.
- Culturing media and conditions (same as test or not): The algae cultures were stored in the laboratory on a solid medium (LC/agar Petri plates) and in a liquid medium with regular sub-culturing (every week).
- Any deformed or abnormal cells observed: Not reported.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test temperature:

22.8ºC - 23.3ºC

pH:

7.4 - 10

Nominal and measured concentrations:

Nominal concentrations (%v/v saturated solution): 0 (control), 15%, 25%, 40%, 62.5% and 100%
Geometric mean measured concentrations: 0 (control), 0.064, 0.065, 0.070, 0.078 and 0.124 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: erlenmeyer flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 300 mL without headspace, 300 mL fill volume.
- Aeration: Not specified.
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 69.7 x 1E+04 cells/mL (average)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes (OECD TG 201 medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the OECD medium was prepared as per the OECD guideline 201.
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of the controls and the test solutions was checked at the beginning and at the end of the test. Temperature inside the algal chamber was recorded several times per day.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Constant illumination
- Light intensity and quality: 5851 Lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: Beckman Coulter Z2 and Malassez counting cell (observation times: 24, 48 and 72 hours)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: serial dilutions from stock saturated solution at geometric factor of 1.6.
- Justification for using less concentrations than requested by guideline: Not applicable; 5 concentrations used for the main test.

- Range finding study
- Test concentrations: 0 (control), 0.1, 1 and 10 mg/L (nominal test concentrations).
- Results used to determine the conditions for the definitive study: In the range finding test the average inhibition growth rate at 72h was 0.1, 3.9, and 6.5% at the tested concentrations of 0.1, 1 and 10 mg/L, respectively. The chemical analyses revealed that test item concentrations were not stable during the test period. Based on these results, the main test was conducted on five concentrations levels obtained from serial dilutions of a saturated stock solution (100 mg/L nominal concentration): 100% - 62.5% - 40.0% - 25% and 15% (expressed as % v/v saturated solution).

Reference substance (positive control):

yes

Remarks:

(potassium dichromate)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.124 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on no significant difference compared to control observed at the highest tested concentration 0.124 mg/L (Statistical significance determined with the Dunnett test (α=5%))

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.124 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: A regression model was not relevant to calculate an ErCX-(0-72h) because inhibition growth rate was only 5.2% at the highest tested concentration 0.124 mg/L.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.124 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: A regression model was not relevant to calculate an ErCX-(0-72h) because inhibition growth rate was only 5.2% at the highest tested concentration 0.124 mg/L.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.124 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: A regression model was not relevant to calculate an EyCX-(0-72h) because the data does not allow it.

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.124 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: A regression model was not relevant to calculate an EyCX-(0-72h) because the data does not allow it.

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Not reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not reported.

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the last algal growth inhibition test performed with the reference item K2Cr2O7 (March 12, 2019) indicates that the algae sensitivity is correct (within the accepted interval of 0.65 to 1.73 mg/L fixed by the International Standard NF EN ISO 8692 from Mai 2012 as mentioned in the OECD 201 Guideline) and in accordance with previous data obtained by the laboratory.
- ErC50 (0-72h) = 0.83 mg/L (0.68 – 1.02)

Reported statistics and error estimates:

For NOEC determination, statistical significance was determined with the Dunnett test (α=5%) from raw data with the scientific software Minitab 17. A regression model was not relevant to calculate an ErCX-(0-72h) because inhibition growth rate was only 5.2% at the highest tested concentration 0.124 mg/L.

Table 1: pH values during the test

Nominaltest item concentration (% V/V)	pH at the start	pH after 72 hours
Control	7.2	9.6
15	7.3	9.7
25	7.4	9.9
40	7.4	10.0
62.5	7.5	9.8
100	7.7	9.6

The pH at final time (72 hours) has increased by more than 1.5, as recommended by OECD 201 guideline. This is due to absence of headspace in closed bottle. pH increase because of the photosynthetic uptake of CO2. However, the algal growth was sufficient in absence of headspace and the validity criteria of the increase biomass in the control during 72 hours was upper than 16 (= 69.7). This deviation had no incidence on study results.

Table 2. Algal growth inhibition

Nominaltest item concentration (% V/V)		[algae] *10^4/mL at 24 hours			[algae] *10^4/mL at 48 hours			[algae] *10^4/mL at 72 hours
		Count.	Average	CV	Count.	Average	CV	Count.	Average	CV
Control	a	4,2	3,9	15.1	12,2	14,4	21,4	60,8	69,7	10,6
	b	3,3			11,5			78,6
	c	4,1			14,0			65,8
	d	3,7			17,6			69,3
	e	3,4			12,3			65,1
	f	4,9			18,8			78,6
15	a	3,4	4,0	13.2	17,5	17,3	11,9	70,0	72,2	10,1
	b	4,4			15,2			66,2
	c	4,2			19,3			80,3
25	a	4,2	3,3	22.5	21,1	14,7	38,1	77,1	62,1	21,1
	b	2,9			11,9			52,8
	c	2,9			11,0			56,4
40	a	3,0	3,2	6.6	20,6	17,9	13,1	66,4	66,9	4,6
	b	3,1			16,7			64,1
	c	3,4			16,4			70,2
62.5	a	4,1	3,9	13.6	15,0	15,2	14,8	67,8	65,4	6,2
	b	4,3			17,6			67,7
	c	3,3			13,1			60,7
100	a	2,8	3,0	6.9	10,2	11,8	12,2	50,2	55,8	14,6
	b	3,1			13,0			65,2
	c	3,2			12,2			52,1

Table 3. Coefficients of variation for section-by-section specific growth rates in the control cultures

Control replicates		Specific growth rate
		0- 24h	Average	CV (%)	24 - 48h	Average	CV (%)	48 - 72h	Average	CV (%)	0 - 72h	Average	CV per replicate (%)	Average CV
Control	a	1,435	1.360	10.9	1,066	1.289	12.6	1,606	1.591	12.3	1,369	1.415	20,1	15.8
	b	1,194			1,248			1,922			1,455		27,9
	c	1,411			1,228			1,548			1,396		11,5
	d	1,308			1,560			1,371			1,413		9,3
	e	1,224			1,286			1,666			1,392		17,2
	f	1,589			1,345			1,431			1,455		8,5

Table 4. Inhibitions of the average specific growth rate in comparison to the Control

Geometric mean measuredtest item concentration		Algae *10^4/mL at 0 hour	Specific  growth rate µ0-72h	Inhibition growth rate (% Ir)	Average inhibition of growth rate (%)	Coefficient of variation	Significance
Control	a	1	1,369	Not applicable		2.5	-
	b		1,455
	c		1,396
	d		1,413
	e		1,392
	f*		1,455
0.064 mg/L	a	1	1,416	-0,1	-0,8	Not required	ns
	b		1,398	1,2
	c		1,462	-3,3
0.065 mg/L	a	1	1,448	-2,4	2,7		ns
	b		1,322	6,5
	c		1,344	5,0
0.070 mg/L	a	1	1,399	1,1	1,0		ns
	b		1,387	2,0
	c		1,417	-0,2
0.078 mg/L	a	1	1,406	0,7	1,5		ns
	b		1,405	0,7
	c		1,369	3,3
0.124 mg/L	a	1	1,305	7,7	5,2		ns
	b		1,392	1,6
	c		1,318	6,9

- : not relevant

ns : not significantly different compared to the Control. Dunnet test, α =0.05

* significantly different compared to the Control, Dunnett test, α=0.05

Table 5. Inhibitions of the yield in comparison to the Control

Geometric mean measuredtest item concentration		[algae] *10^4/mL at 0 hour	[algae] *10^4/mL at 72 hours	Yield (*10^4/ mL)	Average yield (*10^4/mL)	Yield inhibition (Iy) (%)
						Per replicate	Average/ replicate
Control	a	1	60,8	59,8	68,7	Not Applicable
	b		78,6	77,6
	c		65,8	64,8
	d		69,3	68,3
	e		65,1	64,1
	f		78,6	77,6
0.064 mg/L	a	1	70,0	69,0	71,2	-0,4	-3,6
	b		66,2	65,2		5,1
	c		80,3	79,3		-15,4
0.065 mg/L	a	1	77,1	76,1	61,1	-10,8	11,1
	b		52,8	51,8		24,6
	c		56,4	55,4		19,4
0.070 mg/L	a	1	66,4	65,4	65,9	4,8	4,1
	b		64,1	63,1		8,2
	c		70,2	69,2		-0,7
0.078 mg/L	a	1	67,8	66,8	64,4	2,8	6,3
	b		67,7	66,7		2,9
	c		60,7	59,7		13,1
0.124 mg/L	a	1	50,2	49,2	54,8	28,4	20,2
	b		65,2	64,2		6,6
	c		52,1	51,1		25,6

Table 6: Conclusion on the chemical analysis

Nominal test item concentration (% V/V)	Results of the determination of test item analysis (mg/L)*
	0 hour Initial time	24 hours	Deviation T0h/T24h (%)	48 hours	Deviation T0h/T48h (%)	72 hours	Deviation T0h/T72h (%)
Control	< 0.1	< 0.1	NA	< 0.1	NA	< 0.1	NA
15	0.21	< 0.1	NA	< 0.1	NA	< 0.1	NA
25	0.24	< 0.1	NA	< 0.1	NA	< 0.1	NA
40	0.38	< 0.1	NA	< 0.1	NA	< 0.1	NA
62.5	0.73	< 0.1	NA	< 0.1	NA	< 0.1	NA
100	1.49	0.14	-90.6	< 0.1	NA	< 0.1	NA

NA: Non Applicable

* <  0.1 mg/L: LOQ  

According to the results, the measured concentrations of test item in samples were not maintained within ± 20 per cent of the measured initial concentration. Consequently, the test item was not considered as stable in the testing conditions after 24 hours of exposure and during the study. Therefore, and in agreement with the Guidance document OECD nº23, concentrations were expressed in geometric mean measured test item concentrations as calculated in Annex 2 of the OECD nº23. In order to calculate a mean exposure concentration when the substance is lower than the Limit Of Quantification (LOQ), it was used half of limit of quantification (0.1 / 2 =0.05 mg/L).

Table 7: Geometric mean exposure measured concentrations

Nominal test item concentration (%)	Test item analysis (mg/L)				Geometric mean exposure measured concentration* (mg/L)
	0 hour Initial time	24 hours	48 hours	72 hours
Control	< 0.1	< 0.1	< 0.1	< 0.1	NA
15	0.21	< 0.1	< 0.1	< 0.1	0.064
25	0.24	< 0.1	< 0.1	< 0.1	0.065
40	0.38	< 0.1	< 0.1	< 0.1	0.070
62.5	0.73	< 0.1	< 0.1	< 0.1	0.078
100	1.49	0.14	< 0.1	< 0.1	0.124

NA: Non Applicable

*: the number of decimal is required for testing

Validity criteria fulfilled:

yes

Remarks:

(increase of biomass in control during 72h>16 fold; coefficient of variation of the mean specific growth rate among replicates in control (t0-t72)<7%; the mean of the replicate coefficients of variation in section-by-section growth rate in control < 35%)

Conclusions:

The 72-h NOEC of the test substance on Pseudokirchneriella subcapitata was determined to be 0.124 mg/L (value expressed in geometric mean measured concentration taking into account losses of test item during the study).

Executive summary:

An Algae Growth Inhibition Test was performed with Pseudokirchneriella subcapitata green algae on the test substance over a period of 72 h in static conditions according to OECD Guideline 201, following GLP. The test item was found poorly soluble and volatile, then a special solubilisation protocol in the test medium (OECD medium) was carried out before the experimentation, according to the OECD nº23 guidance document on aquatic toxicity testing of difficult substances and mixtures. No vehicle was needed to be used. In a preliminary range finding study the average inhibition growth rate at 72h was 0.1, 3.9, and 6.5% at the tested concentrations of 0.1, 1 and 10 mg/L, respectively. The chemical analyses revealed that test item concentrations were not stable during the test period. Based on these results, the main test was conducted on five concentrations levels obtained from serial dilutions of a saturated stock solution (100 mg/L nominal concentration): 100% - 62.5% - 40.0% - 25% and 15% (expressed as % v/v saturated solution). Analysis of all test concentrations at 24 h intervals was performed during the main test. The results were determined and expressed relative to the geometric mean of the measured concentrations during the test period which were calculated to be 0 (control), 0.064, 0.065, 0.070, 0.078 and 0.124 mg/L (calculated according to OECD nº 23, 15/12/00). The test was conducted under constant illumination (5851 Lux) in closed erlenmeyer flasks filled with 300 mL alga medium and without headspace. 3 replicates were performed for each concentration of the test item, along with six replicates for the negative control group. Test medium alone was used as negative control and a solution of potassium dichromate was used as reference substance. A validated analytical method based on HPLC/UV was used to monitor the concentration of the active ingredient in the test solutions. The validity criteria were successfully fulfilled: In the control the increase of the biomass during 72 hours was determined to be higher than factor of 16. The coefficients of variation of daily section-by-section specific growth rate and of average specific growth rate were determined respectively to be less than 35% and 7% in the control. pH level in the control varied more than 1.5 units at the end of the test. However, the algal growth was sufficient (factor higher than 16) and thus this deviation was considered without incidence on study results. The percent inhibitions in average specific growth rate were -0.8, 2.7, 1.0, 1.5 and 5.2% and the percent inhibitions in yield were -3.6, 11.1, 4.1, 6.3 and 20.2% at the tested geometric mean measured concentrations of 0.064, 0.065, 0.070, 0.078 and 0.124 mg/L respectively, when compared to the negative control. Based on these results, ErC50-(0-72h), ErC10-(0-72h), EyC50-(0-72h) or EyC10-(0-72h) are estimated to be higher than 0.124 mg/L. Thus, the 72 h-NOEC can be determined to be 0.124 mg/L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: ASTM methods (ASTM, 1988)

Version / remarks:

The inhibition concentration (IC50), the concentration at which there was a 50 percent growth inhibition was calculated using a linear interpolation program (Marcus and Holtzman, 1988; Norberg-King, 1988).

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 88, 175, 350 and 700 μg/L
- Sampling time: 0, 24, and 96 hours

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Seventy-five milliliters of test solution were placed in 125 ml volumetric flasks. This was done to minimize headspace. Dilution water was deionized then filtered through a 0.22 micron filter. After this procedure, micro and macronutrients were added to the dilution water.
Stock solutions were also supplied with the necessary nutrients to make concentrations compatible to the dilution water. Five concentrations of stock were used: 100%, 50%, 25%, 12.5% and 0%.
- Test cell concentrations were approximately 1 x10^4 cells/ml. A coulter counter, an electronic particle counter, was used to count cells and determine a mean cell volume
- Controls: Yes, dilution water
- Chemical name of vehicle: water

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
ACCLIMATION
No data

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

no data

Test temperature:

no data

pH:

no data

Dissolved oxygen:

no data

Salinity:

no data

Conductivity:

no data

Nominal and measured concentrations:

Nominal concentrations: 0, 88, 175, 350 and 700 μg/L
Measured concentrations: No available

Details on test conditions:

TEST SYSTEM
- Test vessel: 125 ml volumetric flasks
- Material, size, headspace, fill volume: glass, 125 ml, 75 milliliters of test solution were placed in the flask to minimize headspace.
- Initial cells density: 1x10^4 cells/ml
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

GROWTH MEDIUM
According to ASTM.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised.
- Culture medium different from test medium: No
- Intervals of water quality measurement: 0, 24 and 96 hrs (the 96 hr sample was done only if the chemical was present at the 24 hr sampling)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: A coulter counter, an electronic particle counter, was used to count cells and determine a mean cell volume

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: No
- Test concentrations: 0, 88, 175, 350 and 700 μg/L

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 700 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Growth inhibition

Remarks on result:

other: No significant inhibition observed up to the highest concentration tested

Results with reference substance (positive control):

not applicable

Validity criteria fulfilled:

yes

Conclusions:

The 96h-EC50 of d-alpha pinene to green algae Pseudokirchneriella subcapitata was calculated to be higher than 700 μg/L based on growth inhibition.

Executive summary:

A toxicity test was conducted on d-alpha pinene using green algae (Selenastrum capricornutum) according to ASTM methods for conducting static 96 hour toxicity tests with microalgae (ASTM, 1988). Deionised water was used to formulate the test solutions. The test was performed in nominal concentrations of 0, 88, 175, 350 and 700 μg/L. Flasks were set up in replicates of four at each concentration and shaken continuously. GC analysis was performed at 0, 24 and 96 hrs. No significant effects based on growth inhibition were observed at any concentration tested. Thus, the 96h-NOEC and 96h-EC50 were found to be higher than 700 μg/L.

Description of key information

Key study. Test method according to OECD 201, GLP study. The 72-h NOEC of the test substance on Pseudokirchneriella subcapitata was determined to be 0.124 mg/L (value expressed in geometric mean measured concentration taking into account losses of test item during the study).

Supporting study: Test performed according to ASTM methods. The 96h-EC50 of d-alpha pinene to green algae Pseudokirchneriella subcapitata was calculated to be higher than 700 μg/L based on growth inhibition.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.124 mg/L

Additional information

Key study: An Algae Growth Inhibition Test was performed with Pseudokirchneriella subcapitata green algae on the test substance over a period of 72 h in static conditions according to OECD Guideline 201, following GLP. The test item was found poorly soluble and volatile, then a special solubilisation protocol in the test medium (OECD medium) was carried out before the experimentation, according to the OECD nº23 guidance document on aquatic toxicity testing of difficult substances and mixtures. No vehicle was needed to be used. In a preliminary range finding study the average inhibition growth rate at 72h was 0.1, 3.9, and 6.5% at the tested concentrations of 0.1, 1 and 10 mg/L, respectively. The chemical analyses revealed that test item concentrations were not stable during the test period. Based on these results, the main test was conducted on five concentrations levels obtained from serial dilutions of a saturated stock solution (100 mg/L nominal concentration): 100% - 62.5% - 40.0% - 25% and 15% (expressed as % v/v saturated solution). Analysis of all test concentrations at 24 h intervals was performed during the main test. The results were determined and expressed relative to the geometric mean of the measured concentrations during the test period which were calculated to be 0 (control), 0.064, 0.065, 0.070, 0.078 and 0.124 mg/L (calculated according to OECD nº 23, 15/12/00). All validity criteria were successfully fulfilled. pH level in the control varied more than 1.5 units at the end of the test. However, the algal growth was sufficient (factor higher than 16) and thus this deviation was considered without incidence on study results. The percent inhibitions in average specific growth rate were -0.8, 2.7, 1.0, 1.5 and 5.2% and the percent inhibitions in yield were -3.6, 11.1, 4.1, 6.3 and 20.2% at the tested geometric mean measured concentrations of 0.064, 0.065, 0.070, 0.078 and 0.124 mg/L respectively, when compared to the negative control. Based on these results, ErC50-(0-72h), ErC10-(0-72h), EyC50-(0-72h) or EyC10-(0-72h) are estimated to be higher than 0.124 mg/L. Thus, the 72 h-NOEC can be determined to be 0.124 mg/L.

Supporting study: A toxicity test was conducted on d-alpha pinene using green algae (Selenastrum capricornutum) according to ASTM methods for conducting static 96 hour toxicity tests with microalgae (ASTM, 1988). Deionised water was used to formulate the test solutions. The test was performed in nominal concentrations of 0, 88, 175, 350 and 700 μg/L. Flasks were set up in replicates of four at each concentration and shaken continuously. GC analysis was performed at 0, 24 and 96 hrs. No significant effects based on growth inhibition were observed at any concentration tested. Thus, the 96h-NOEC and 96h-EC50 were found to be higher than 700 μg/L.