p-mentha-1,3-diene

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:

March-April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: Alpha-terpinene
Batch/Lot Number: 83100001
CAS Number: 99-86-5
EC Number: 202-795-1
Scymaris Reference Number: 1027.005
Purity: 92.7%
Water Solubility: Immiscible
Appearance: Clear colourless liquid
Expiry Date: 11 May 2018
Storage Conditions: Room temperature

Analytical monitoring:

yes

Remarks:

Measured using GCMS

Details on sampling:

The concentrations of Alpha-terpinene in the test solutions detailed below were measured at 0 and 72 hours for each test concentration using GCMS.

Samples for analysis at 0 hours were taken from the excess test solutions of the control and each test concentration and at 72 hours from a single test replicate solution (containing algae) of each exposure concentration. All samples taken for analysis at 72 hours, which contain algae, were analysed post centrifugation. All other samples were analysed directly.

Vehicle:

yes

Remarks:

Use of solvent - DMF (dimethylformamide) solvent

Details on test solutions:

The range finding study was performed using DMF (dimethylformamide) as a solvent, with the following test solutions: control, solvent control and nominal concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L.

In the definitive test, the study was performed usign DMF as a solvent and the following test concentrations control, solvent control and nominal concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L. These were based on the findings of the range finding study.

Test organisms (species):

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

Details on test organisms:

The test organism was the green alga Pseudokirchneriella subcapitata (strain CCAP 278/4) from laboratory cultures. Stock cultures were maintained under axenic conditions.

A culture of the alga in the exponential growth phase was used as the inoculum. The growth of the parent culture was measured to determine if the cell density increase is within an acceptable range (typically by a factor of 16 times in a 72 hour period, equivalent to a specific growth rate of 0.92 per day). Each test vessel was inoculated individually, with an addition of the inoculum not exceeding 2% by volume, such that the initial cell density is 0.5 (± 0.1) ×104cells/mL. This density was confirmed by the addition of the selected volume of inoculum culture to one 100 mL volume of ISOTON II which was counted as described above. The parent culture was examined under a microscope to verify a normal healthy culture was being tested. The inoculum was cultured in the medium and under the conditions described for the test.

Test type:

static

Water media type:

freshwater

Remarks:

AAP-medium

Total exposure duration:

72 h

Test temperature:

21-24°C ± 2°C

Nominal and measured concentrations:

Range finding study
Chemistry showed that at 0 hours we achieved 71-110% of nominal and at 72 hours this dropped to 5-9% of nominal.

Definitive test
Final geometric mean concentrations in the definitive test were 25 to 53% of the nominal concentrations.

Details on test conditions:

The test vessels were glass conical flasks of 250 mL nominal capacity, with foam closures, containing 100 mL of test solution. The cultures were incubated at 21-24°C controlled at ± 2°C, under continuous illumination of approximately 6000 lux, with orbital shaking at 160 ± 10 rpm. The positions of test vessels in the incubator were randomised daily. One blank vessel (without algal inoculum) was incubated concurrently for the control and each test concentration. Blanks will not be randomised daily. The algal cell densities of the inoculum and test cultures were determined by electronic particle counting using a Coulter counter. Particles were counted between a lower and upper threshold equivalent spherical diameter of approximately 2.3 and 5.0 µm respectively. Each replicate test vessel was inoculated individually to give a nominal initial cell density of 0.5 (± 0.1) × 104 cells/mL. After 24, 48 and 72 hours, samples were removed from each blank and test vessel and the particle cell density counted. The appropriate blank particle count was subtracted from that of the test culture to obtain the cell density.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 3.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

In the definitive test, there was no significant effect on the yield at any concentration when compared to the control or solvent control. Measured concentrations in the test solutions were 50-107% of the nominal at 0 hours but all dropped to below the limit of detection by 72 hours. As such, the geometric mean measured test concentrations were 0, 0.16, 0.47, 1.1, 2.7 and 3.7 mg/l (equivalent to nominal concentrations, 0, 0.625, 1.25, 2.5, 5.0 and 10 mg/l, respectively). Therefore, a NOEC of 3.7 mg/l and a LOEC of >3.7 mg/l (based on geometric mean measured concentrations) have been reported for this study.

Table 1. Range finding study yield results

Nominal concentration (mg/L)	Mean Biomass 72 hours (cells/ml×10^4)	Percentage of solvent control (%)
Control	42.97	92
Solvent Control	46.77	-
0.625	45.37	97
1.25	45.30	97
2.5	46.10	99
5.0	51.07	109
10	47.30	101

72 hour microscopic observations:

Control, solvent control and all test concentrations: Normal

Table 2. Range finding study analytical results

Nominal concentration (mg/L)	0 hour measured concentration (mg/L)	0 hour measured conc. (% of nominal)	72 hour measured concentration (mg/L)	72 hour measured conc. (% of nominal)	Mean measured concentration (Geometric mean) (mg/L)	Mean measured concentration (Geometric mean) (% of nominal)
Control	<LOQ	-	<LOQ	-	-	-
Solvent Control	<LOQ	-	<LOQ	-	-	-
0.625	0.69	110	0.057	9	0.20	32
1.25	1.1	90	0.11	9	0.35	28
2.5	2.5	100	0.11	5	0.52	21
5.0	4.8	96	0.23	5	1.1	22
10	7.1	71	0.57	6	2.0	20

LOQ varied from 0.01 to 1.00 mg/L due to different dilution factors (increasing with concentration).

If values are <LOQ then zero was used in calculation of the mean.

Table 3. Definitive test yield results

Nominal concentration (mg/L)	Mean Yield 72 hours (cells/mLx×10^4)	Percentage of solvent control (%)
Control	26.0	97
Solvent Control	26.9	-
0.625	29.2	109
1.25	27.0	100
2.5	27.5	102
5.0	29.9	111
10	27.0	100

 

Table 4. Definite test analytical results.

Nominal concentration (mg/L)	0 hour measured conc. (mg/L)	0 hour measured conc. (% of nominal)	72 hour measured conc. (mg/L)	72 hour measured conc. (% of nominal)	Mean measured conc. (arithmetic) (mg/L)	Mean measured conc. (% of nominal)
Control	<LOQ	-	<LOQ	-	-	-
Solvent Control	<LOQ	-	<LOQ	-	-	-
0.625	0.31	50	<LOQ	-	0.16	25
1.25	0.94	76	<LOQ	-	0.47	38
2.5	2.1	85	<LOQ	-	1.1	43
5.0	5.3	107	<LOQ	-	2.7	53
10	7.4	74	<LOQ	-	3.7	37

LOQ varied from 0.01 to 1.00 mg/L due to different dilution factors (increasing with concentration).

If values are <LOQ then zero was used in calculation of the mean.

Validity criteria fulfilled:

not specified

Conclusions:

In the definitive test, there was no significant effect on the yield at any concentration when compared to the control or solvent control. Measured concentrations in the test solutions were 50-107% of the nominal at 0 hours but all dropped to below the limit of detection by 72 hours. As such, the geometric mean measured test concentrations were 0, 0.16, 0.47, 1.1, 2.7 and 3.7 mg/l (equivalent to nominal concentrations, 0, 0.625, 1.25, 2.5, 5.0 and 10 mg/l, respectively). Therefore, a NOEC of 3.7 mg/l and a LOEC of >3.7 mg/l (based on geometric mean measured concentrations) have been reported for this study.

Executive summary:

The effects of Alpha-terpinene on the growth of alga Pseudokirchneriella subcapitata was determined over 72 hours, according to OECD guideline 201. A NOEC of 3.7 mg/l and a LOEC of >3.7 mg/l (based on geometric mean measured concentrations) have been reported for this study.

Description of key information

Key study: Test method according to OECD 201, GLP study. The effects of Alpha-terpinene on the growth of alga Pseudokirchneriella subcapitata was determined over 72 hours. A NOEC of 3.7 mg/l and a LOEC of >3.7 mg/l (based on geometric mean measured concentrations) have been reported for this study.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

3.7 mg/L