Rectified Hydrocarbons by-products from synthetic process of Turpentine and acid, alcohols fraction

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 December 2018 – 11 January 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 6.7, 11.5, 19.5, 33.0, 56.2 and 89.7 mg/L (nominal loading rates)
- Sampling method: During Range finding study and main study, TOC was analyzed on day 0 (fresh) and on day 1 (spent) from samples of all the loading rates. The test media in the aquarium was mixed thoroughly before the collection of sample.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
The test item is a complex mixture (UVCB substance) which was found to be partially miscible in test medium, i.e. reverse osmosis water (tap water) according to a lab internal method. Therefore, the Fish Test was carried out with aqueous extracts (WAF = Water Accommodated Fraction) at various loading rates of the test item in test medium.
In order to determine the optimum mixing duration to be used for WAF preparation, a water accommodated fraction (WAF) equilibration trail was performed prior to exposure: One individual WAF was prepared at each of three loading levels 1.0, 50.0 and 100 mg/L. At 2, 4, 6 and 24 hours after initiation of mixing, mixing was stopped at respective time points and the solutions were allowed to settle for one hour. A sample of WAF was removed from each loading level and mixing was resumed. The concentration of organic carbon that had solubilized into the WAF from the test item was measured using the TOC analyzer described above.
The test item equilibrium was attained at 24 hour mixing according to the equilibration trail, hence mixing for 24 hours was followed during the test media preparation.
Test media preparation: For every test item concentration and every renewal period (semi-static test), individual WAF was prepared by adding the appropriate amount of test item to the test medium. Each mixing vessel was filled to achieve minimal headspace given the constraints of the vessels and closed with foil-covered stoppers. The mixture was stirred for 24 h (based on equilibration trail) using a magnetic stirrer at minimum of 100 rpm. The mixtures were allowed to settle and equilibrate to test temperature (before removing the test solution). The aqueous portions of WAFs were collected using steri-pipette and transferred to test vessels.
- Controls: reverse osmosis water (tap water) only.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test substance was not fully soluble at the tested loading rate. However the method of preparing the WAFs was designed to mimimise exposure to undissolved test material.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Cyprinus carpio (common carp)
- Source: Fisheries Research and Information Center (Inland), Hebbal, Bangalore, Karnataka, India
- Length at study initiation (length definition, mean, range and SD): mean (for every group) = 3.0 - 3.2 cm; range (all groups) = 2.9 to 3.4 cm; SD (for every group) = 0.1 - 0.2

ACCLIMATION
- Acclimation period: 7 days
- Acclimation conditions (same as test or not): same as test
- Type and amount of food during acclimation: commercial feed pellets (Kijaro Grow)
- Feeding frequency during acclimation: ad libitum daily
- Health during acclimation (any mortality observed): no mortality was observed


FEEDING DURING TEST . None

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

216 to 224 mg/L of CaCO3

Test temperature:

22.1 to 23.1ºC

pH:

6.70 to 7.12

Dissolved oxygen:

78.2 to 89.8%

Nominal and measured concentrations:

Nominal loading rates: 0 (control), 6.7, 11.5, 19.5, 33.0, 56.2 and 89.7 mg/L WAF

Details on test conditions:

TEST SYSTEM
- Test vessel: aquarium
- Material, size, headspace, fill volume: glass, 50 L, 25 L (fill volume).
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): 24 h
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): N/A
- Biomass loading rate: 0.6 g/L (7 fish / aquarium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis water (tap water)
- Intervals of water quality measurement: Temperature, pH and dissolved Oxygen were measured every 24 h in old and fresh solutions. Total hardness was analyzed daily in fresh solutions.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 12 hour light and 12 hour darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Fish was observed for signs of toxicity (behavioural and morphological responses) and mortality at 3, 6, 24, 48, 72 and 96 hour (± 20 min) of exposure. Fish was considered dead if no visible movements (gill movements) were observed and no response was produced upon touching of the caudal peduncle. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). Dead fish was removed when observed and the mortality was recorded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.7
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study
- Test concentrations: 0.90, 8.97, 44.86, 67.28 and 89.7 mg/L (nominal loading rates) along with control.
- Results used to determine the conditions for the definitive study: Mortalities of 14.3, 57.1, 71.4 and 100.0% were observed at the tested nominal loading rates of 8.97, 44.86, 67.28 and 89.7 mg/L. No mortalities were observed in the control group and at the nominal loading rate of 0.90 mg/L.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

34.67 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(water-accommodated fraction loading rate)

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence limits: 30.05- 40.1 mg/L.

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

6.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(water-accommodated fraction loading rate)

Basis for effect:

behaviour

Duration:

96 h

Dose descriptor:

LOELR

Effect conc.:

11.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(water-accommodated fraction loading rate)

Basis for effect:

behaviour

Details on results:

- Behavioural abnormalities:
- Abnormal responses: Clinical sign of easy catching was observed at the nominal loading rate of 11.5 mg/L during the 96 hour exposure period. Clinical sign of lethargy and easy catching was observed at the nominal loading rate of 19.5, 33.0, 56.2 and 89.7 mg/L during the 96 hour exposure period.

Reported statistics and error estimates:

The acute Median Lethal Loading rate (LL50) of Pine oil 50% was evaluated by using the Probit analysis (Finney, 1971) with 95% confidence limits based on the nature of the mortality data. The LL50 values were evaluated with fiducial limits and graph was plotted with percent mortality against log concentration for recommended exposure periods of 96 hours.

Sublethal observations / clinical signs:

Table 1. Summary of mortality and 96 hour % mortality during range finding study

Group	Nominal Loading Rates (mg/L)	No. of Fish/Group at Start	Mortality (No.) Observed at						96 hours Percent Mortality
			3 h (±20 Mins)	6 h (±20 Mins)	24 h (±20 Mins)	48 h (±20 Mins)	72 h (±20 Mins)	96 h (±20 Mins)
G1	0.0	7	0	0	0	0	0	0	0
G2	0.90	7	0	0	0	0	0	0	0.0
G3	8.97	7	0	0	0	0	1	0	14.3
G4	44.86	7	0	0	1	1	1	1	57.1
G5	67.28	7	0	0	1	2	2	0	71.4
G6	89.7	7	0	2	1	1	2	1	100.0

Table 2. Summary of clinical signs during main study

Group	Nominal Loading Rates (mg/L)	No. of Fish/Group at Start	Clinical Signs of Toxicity at
			3 h (±20 Mins)	6 h (±20 Mins)	24 h (±20 Mins)	48 h (±20 Mins)	72 h (±20 Mins)	96 h (±20 Mins)
G1	0.0	7	1(7)	1(7)	1(7)	1(7)	1(7)	1(7)
G2	6.7	7	1(7)	1(7)	1(7)	1(7)	1(7)	1(7)
G3	11.5	7	1(7)	1(7)	1(7)	1(5), 10(2)	1(5), 10(1)	1(5), 10(1)
G4	19.5	7	1(7)	1(7)	1(5), 3(1), 10(1)	1(4), 3(1), 10(1)	1(4), 3(1)	1(4), 3(1)
G5	33.0	7	1(7)	1(5), 10(2)	1(5), 10(1)	1(3), 3(1) 10(1)	1(3), 3(1)	1(3), 3(1)
G6	56.2	7	1(7)	1(3), 10(2), 3(2)	1(3), 10(1), 3(1)	1(2),10(1), 3(1)	10(2), 3(1)	10(1), 3(1)
G7	89.7	7	1(3), 3(1), 10(1)	1(1), 3(1), 10(1)	10(1)	-	-	-

1: Normal; 10: Easy catching; 3: Lethargy

Table 3. Summary of mortality and 96 hour % mortality during main study

Group	Nominal Loading Rates (mg/L)	No. of Fish/Group at Start	Mortality (No.) Observed at						96 hours Percent Mortality	P- Value
			3 h (±20 Mins)	6 h (±20 Mins)	24 h (±20 Mins)	48 h (±20 Mins)	72 h (±20 Mins)	96 h (±20 Mins)
G1	0.0	7	0	0	0	0	0	0	0	-
G2	6.7	7	0	0	0	0	0	0	0	-
G3	11.5	7	0	0	0	0	1	0	14.3	1.0
G4	19.5	7	0	0	0	1	1	0	28.6	0.4615
G5	33.0	7	0	0	1	1	1	0	42.9	0.1923
G6	56.2	7	0	0	2	1	1	1	71.4	*0.0210
G7	89.7	7	2	2	2	1	-	-	100.0	*0.0006

*: Statistical significance of >0.05

Table 4. Summary of total organic carbon (TOC) analysis during main study

Nominal Loading rate (mg/L)	TOC During Exposure (mg)
	Day 0 (Fresh)	Day 1 (Aged)	Recovery (%)
0.0	-	-	-
6.7	3.8940	3.4120	87.62
11.5	4.4620	4.0630	91.06
19.5	9.949	6.4980	65.31
33.0	13.530	11.3500	83.89
56.2	21.700	17.850	82.26
89.7	31.250	27.1500	86.88

Table 5. Individual fish length during main study

Group	Nominal Loading Rates (mg/L)	Fish Length (cm)							Mean	SD	Cummulative Weight (g)	Loading Rate (g/L)
G1	0.0	3.1	3.1	3.0	3.2	3.1	3.2	3.1	3.1	0.1	14.6	0.60
G2	6.7	3.2	3.1	3.0	3.1	3.2	3.1	3.2	3.1	0.1	14.5
G3	11.5	2.9	3.1	3.0	2.9	3.1	3.0	3.2	3.0	0.1	14.1
G4	19.5	3.3	3.1	3.4	3.0	3.1	2.9	3.0	3.1	0.2	14.6
G5	33.0	3.2	3.0	3.1	3.4	3.1	3.3	3.2	3.2	0.1	14.2
G6	56.2	3.2	3.0	3.0	2.9	3.0	3.1	3.1	3.0	0.1	14.7
G7	89.7	3.1	3.2	3.1	3.4	3.3	3.4	3.2	3.2	0.1	15.1
	Min	2.9
	Max	3.4

Table 6. Physico-chemical parameters of test media during main study

Dissolved oxygen (%)

Group	Nominal Loading Rates (mg/L)	0 to 24 h		24 to 48 h		48 to 72 h		72 to 96 h
		Fresh 0 h	Spent 24 h	Fresh 24	Spent 48 h	Fresh 48 h	Spent 72 h	Fresh 72 h	Spent 96 h
G1	0.0	89.0	86.8	89.6	85.2	89.8	83.6	86.8	82.4
G2	6.7	87.6	84.1	88.4	87.6	86.4	84.2	81.4	80.3
G3	11.5	87.8	85.3	83.8	80.2	89.3	85.8	82.3	78.2
G4	19.5	88.2	82.6	85.4	82.2	84.3	81.4	85.1	81.4
G5	33.0	89.1	83.9	86.2	81.3	86.8	83.1	86.3	80.8
G6	56.2	87.6	84.8	81.8	80	84.2	80.6	82.3	79.4
G7	89.7	86.8	85.2	80.8	79.6	-	-	-	-
Min		78.2
Max		89.8

Temperature (ºC)

Group	Nominal Loading Rates (mg/L)	0 to 24 h		24 to 48 h		48 to 72 h		72 to 96 h
		Fresh 0 h	Spent 24 h	Fresh 24	Spent 48 h	Fresh 48 h	Spent 72 h	Fresh 72 h	Spent 96 h
G1	0.0	22.1	22.6	22.8	22.1	22.4	22.3	22.9	22.4
G2	6.7	22.6	22.4	22.9	22.3	23.1	22.4	22.6	22.9
G3	11.5	22.5	22.6	22.6	22.4	22.9	22.6	22.9	22.3
G4	19.5	22.4	22.3	22.4	22.2	22.8	22.4	22.2	22.4
G5	33.0	22.3	22.4	22.9	22.6	22.9	22.9	22.6	22.3
G6	56.2	22.6	22.9	22.8	22.7	23.0	22.8	22.9	22.6
G7	89.7	22.2	22.3	22.7	22.3	-	-	-	-
Min		22.1
Max		23.1

pH

Group	Nominal Loading Rates (mg/L)	0 to 24 h		24 to 48 h		48 to 72 h		72 to 96 h
		Fresh 0 h	Spent 24 h	Fresh 24	Spent 48 h	Fresh 48 h	Spent 72 h	Fresh 72 h	Spent 96 h
G1	0.0	7.00	7.08	6.92	6.99	7.02	6.98	7.12	6.92
G2	6.7	6.91	6.92	6.89	6.86	6.85	6.82	6.89	6.82
G3	11.5	6.92	6.89	6.85	6.82	6.74	6.76	6.82	6.80
G4	19.5	6.93	6.90	6.86	6.84	6.73	6.75	6.79	6.82
G5	33.0	6.89	6.86	6.84	6.80	6.75	6.72	6.76	6.74
G6	56.2	6.88	6.80	6.82	6.81	6.71	6.74	6.73	6.70
G7	89.7	6.86	6.83	6.83	6.82	-	-	-	-
Min		6.70
Max		7.12

Total Hardness (as mg CaCO3/L)

Day	0	1	2	3
Total Hardness	220	220	224	216

Validity criteria fulfilled:

yes

Remarks:

(mortality in control was 0%; constant conditions were maintained; the dissolved oxygen concentration was within the acceptable range; TOC measurements for each nominal loading rate were mostly maintained within ± 20% during a renewal period).

Conclusions:

In a short-term toxicity test to Cyprinus carpio (common carp), the 96h-LL50 of the test substance was found to be 34.67 mg/L.

Executive summary:

An acute aquatic toxicity study with Cyprinus carpio (common carp) was conducted on the test item PINE OIL 50% according to OECD guideline 203, following GLP. The test item is a UVCB substance which was found to be partially miscible in reverse osmosis water (tap water) which was used as test medium. Therefore, the test was carried out in semi-static conditions with aqueous extracts (WAF = Water Accommodated Fraction) at various loading rates of the test item in the test medium. Based on a preliminary WAF equilibration trail, individual WAF solutions for every test item concentration and every renewal period were prepared in closed vessels with minimal headspace and stirred for 24 h. Then, the mixtures were allowed to settle and equilibrate to test temperature and aqueous portions of WAFs were transferred to the test vessels. A range finding study was conducted for 96 h with nominal loading rates of 0.90, 8.97, 44.86, 67.28 and 89.7 mg/L along with control group. Mortalities of 14.3, 57.1, 71.4 and 100.0% were observed at 8.97, 44.86, 67.28 and 89.7 mg/L. No mortalities were observed in the control group and at 0.90 mg/L. Based on these results, the main test was conducted for 96 h at nominal loading rates of 6.7, 11.5, 19.5, 33.0, 56.2 and 89.7 mg/L along with control group. No clinical sign of toxicity was observed in the control group and at 6.7 mg/L nominal loading rate. Clinical sign of easy catching was observed at 11.5 mg/L. Lethargy and easy catching were observed at 19.5, 33.0, 56.2 and 89.7 mg/L. Mortalities of 14.3, 28.6, 42.9, 71.4 and 100.0% were observed at 11.5, 19.5, 33.0, 56.2 and 89.7 mg/L. No mortalities were observed in the control group and at nominal loading rate of 6.7 mg/L. TOC measurements for each nominal loading rate were mostly maintained within ± 20% during a renewal period. Mortality in the control group was 0% and dissolved oxygen concentration in the test media was above 78.2% of the air saturation during main test. Based on these results, the 96 hours No Observed Effect Loading rate (NOELR) and Lowest Observed Effect Loading rate (LOELR) is 6.7 and 11.5 mg/L respectively, and the acute median lethal loading rate (LL50) is 34.67 mg/L (95% confidence limits: 30.05- 40.1 mg/L).

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

September, 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Principles of method if other than guideline:

- Principle of test: The test was undertaken in accordance with the protocol outlined by Wan et al. (1990; 1991), and by Environment Canada (1990a; 1990b).

Environment Canada (1990a) Biological Test Method: Acute Lethality Test Using Rainbow Trout, Environmental Protection Series. Report EPS 1/RM/9, July 1990, amended May 1996. Ottawa, Ontario, Canada.
Environment Canada (1990b) Reference Method for Determining Acute Lethality of Effluents to Rainbow Trout, Reference Method. Environmental Protection Series. Report EPS 1/RM/13, July 1990, amended May 1996. Ottawa, Ontario, Canada

GLP compliance:

not specified

Remarks:

(Information not provided in the study)

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
- Sampling method: a 10 mL pipette was used to collect 10 x 10 mL sub-samples to make up 100 mL sample at each sampling time. These sub-samples were drawn randomly from various locations and depths of the test vessel.
- Sample storage conditions before analysis: Water samples collected were stored in 200 mL amber heat treated glass bottles with aluminum foil lined caps and kept at 4ºC.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Emulsifier RSB/181/5

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Source: Spring Valley Trout Farm, British Columbia
- Age at study initiation: 2-3 months

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

104 mg CaCO3/L (SE=1)

Test temperature:

15ºC (SE=1)

pH:

7.79 (SE=0.01)

Conductivity:

479 µmhos/cm (SE=1)

Nominal and measured concentrations:

Not specified. 5 nominal concentrations below 100 ppm were selected for the test chemical using a Logarithmic Series of Concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 20 L fill volume
- Aeration: Oil-free air was delivered to the test concentrations at a rate of 6.5 ± 1 mL/min/L by means of disposable borosilicate glass Pasteur pipettes.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per vehicle control (replicates): 3
- Biomass loading rate: 0.4 (0.3 - 0.5) g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Fresh ground water with a mean hardness of 95 mg/L CaCO3 from the Pacific Environmental Science Centre (PESC) well in North Vancouver, British Columbia, Canada.
- Total organic carbon: 12.9 mg/L (SE=0.1)
- Pesticides: No residues of organochlorine, organophosphorus or nitrogen containing pesticides were detected in the water samples
- Chlorine: 96.3 mg/L (SE=0.4)
- Alkalinity: 56.1 mg/L (SE=0.1)
- Ca: 35.2 mg/L (SE=0.3)
- Culture medium different from test medium: No
- Intervals of water quality measurement: 5 water samples were taking during september 1997.

OTHER TEST CONDITIONS
- Photoperiod: 16:8 LD with 15 minute dawn/dusk simulation

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Cumulative fish mortality was recorded at 24, 48, 72, and 96-h time intervals.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

6.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Loge (mean) = 1.89

Details on results:

- Behavioural abnormalities:
- Mortality of control: LC50-96 h = 18.2 mg/L
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: There was a chemical loss in all vessels with and without fish. This loss of test chemical in water samples may be attributed to volatilization during
the initial aeration process (Doudoroff et al., 1951; Wan et al., 1990) and glass adsorption (Sharom and Solomon, 1981; Wan et al., 1991).

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

LC50 was calculated using the “Lethal” computer program developed by Stephen (1977, revised in 1983).

Sublethal observations / clinical signs:

Table 1: Toxicity of alpha terpineol

 

Hour	LC50 (mg/L)-rainbow trout
24	6.7
48	6.7
72	6.6
96	6.6

Table 2: Toxicity of emulsifier

Hour	LC50 (mg/L)-rainbow trout
24	21.5
48	18.9
72	18.3
96	18.2

 

Validity criteria fulfilled:

not specified

Conclusions:

The 96h-LC50 of alpha terpineol to rainbow trout (Oncorhynchus mykiss) was calculated to be 6.6 mg/L.

Executive summary:

In an acute aquatic toxicity study performed accordingly to the protocol outlined by Wan et al. (1990; 1991) and by Environment Canada (1990), groups (10/dose) of rainbow trout (Oncorhynchus mykiss) were exposed for 96 hours to alpha terpineol diluted in 10% emulsifier at 5 nominal concentrations (up to 100 ppm in logarithmic series) under static conditions. Each test concentration was replicated three times. The emulsifier was tested in the same conditions. Quality of dilution water before conducting the test as well as content of test matearial during test were analysed. Mortality was observed at 24, 48, 72 and 96 hours. There was a chemical loss in all vessels with and without fish. The 96 hour LC50 of the test material was calculated to be 6.6 mg/L.

 

 

 

Reference 3

Endpoint:

short-term toxicity to fish

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

Remarks:

Study conducted similarly to OECD guideline 203 but without GLP compliance and with minor deviations (no data on acclimation period). The report is sufficiently well documented.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

No data on acclimation period

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 1060, 2120, 3190, 4250 and 5310 μg/L
- Sampling time: 0, 24, 48, 72 and 96 hours

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions was provided as liquid/liquid saturation units by adding excess of test chemical in 14 L of Lake Superior water
- Controls: Dilution water
- Chemical name of vehicle: water
- Concentration of vehicle in test medium: Stock (5310 µg/L liq-liq equil.)

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnows
- Source: ERL-Duluth, USA
- Age at study initiation: 30 days
- Length at study initiation: 16.8 mm (SD=2.7)
- Weight at study initiation: 57 mg (SD=30)

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

44.9 mg CaCO3/L (SD=0.2)

Test temperature:

25.2ºC (SD=0.6)

pH:

7.5 (SD=0.3)

Dissolved oxygen:

6.1 mg O2/L (SD=0.9)

Salinity:

42.7 mg CaCO3/L (SD=0.7)

Nominal and measured concentrations:

Nominal concentrations: 0, 1060, 2120, 3190, 4250 and 5310 μg/L
Average measured concentrations: <195, 1050, 2090, 2720, 3640 and 4820 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Six crystallizing dishes were used as test chambers in the electronic diluter with tank volumes of 200 mL; test solution depth: 4-5 cm
- Type (delete if not applicable): Closed
- Type of flow-through: Electronic diluter
- Renewal rate of test solution (frequency/flow rate): Flow rates of 21 mL/min to each tank produced toxicant concentrations of 1050-4820 μg/L
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): One
- No. of vessels per vehicle control (replicates): One
- Biomass loading rate: 2.85 g/L

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: Lake Superior water
- Preparation of dilution water: Water was passed through sand filters, ultraviolet sterilizers and 55 µm fibre filter. Test waters were then aerated and heated at 25 ± 1 °C
- Intervals of water quality measurement: Tank temperatures (ºC), dissolved oxygen (mg/L) and pH were measured daily in each of the test chambers where test organisms were still alive

OTHER TEST CONDITIONS
- Photoperiod: 16 hour/day light cycle; cool white fluorescent light
- Light intensity: 12-28 lumens

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Behavior and morphological aberrations were observed at 0, 24, 48, 72 and 96 hours

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

3 150 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL: 2920-3400 μg/L

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

1 480 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: Affected fish swam with a corkscrew motion, were hypoactive and underreactive to external stimuli and were darkly colored. They also had convulsions and lost equilibrium prior to death.
- Mortality (96 hour) at <195, 1050, 2090, 2720, 3640 and 4820 μg/L (measured) were 0, 0, 0, 10, 90 and 100%, respectively.
- Effect (96 hour) at <195, 1050, 2090, 2720, 3640 and 4820 μg/L (measured) were observed in 0/10, 0/10, 10/10, 10/10, 10/10 and 10/10 fish respectively.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

- LC50 or EC50 values were calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber Method (Hamilton et al., 1977).
- Mean toxicant concentrations used in the calculations were corrected for analytical spike recoveries

Sublethal observations / clinical signs:

Table 1: Mortalities

 

Hour	Control (0 µg/L)	A (1060 µg/L)	B (2120 µg/L)	C (3190 µg/L)	D (4250 µg/L)	E (5310 µg/L)
Initial	9	10	10	10	10	10
24	0	0	0	1	7	10
48	0	0	0	1	7	10
72	0	0	0	1	9	10
96	0	0	0	1	9	10

Table 2: Effects

Hour	Control (0 µg/L)	A (1060 µg/L)	B (2120 µg/L)	C (3190 µg/L)	D (4250 µg/L)	E (5310 µg/L)
Initial	9	10	10	10	10	10
24	0	0	0	10	10	10
48	0	0	10	10	10	10
72	0	0	10	10	10	10
96	0	0	10	10	10	10

 

Validity criteria fulfilled:

yes

Remarks:

(mortality in control < 10%; flow-through test conditions; dissolved O2 concentration > 60% of air saturation value; results based on measured concentrations)

Conclusions:

The 96h-LC50 of alpha terpinene to fathead minnow (Pimephales promelas) was calculated to be 3150 (2920 -3400) μg/L.

Executive summary:

In an acute aquatic toxicity study performed similarly to OECD guideline 203, groups (10/dose) of fathead minnows (Pimephales promelas) were exposed to alpha terpinene at concentrations of <195, 1050, 2090, 2720, 3640 and 4820 μg/L (measured) for 96 hours under flow-through conditions. Mortality, behaviour and morphological aberrations were observed at 24, 48, 72 and 96 hours. All validity criteria were fulfilled. Mortalities (96 hour) at <195, 1050, 2090, 2720, 3640 and 4820 μg/L (measured) were 0, 0, 0, 10, 90 and 100%, respectively. Treatment-related effects (96 hour) at <195, 1050, 2090, 2720, 3640 and 4820 μg/L (measured) were observed in 0/10, 0/10, 10/10, 10/10, 10/10 and 10/10 fish respectively. The 96 hour LC50, EC50 and NOEC of alpha terpinene to fathead minnow (Pimephales promelas) were calculated to be 3150 (2920 -3400) μg/L, 1480 μg/L and 1050 μg/L, respectively.

 

 

 

Reference 4

Endpoint:

short-term toxicity to fish

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

Remarks:

Study conducted similarly to OECD guideline 203 but without GLP compliance and with minor deviations (no data on acclimation period). The report is sufficiently well documented.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

No data on acclimation period

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 130, 260, 390, 520 and 650 μg/L
- Sampling time: 0, 24, 48, 72 and 96 hours

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions was provided as liquid/liquid saturation units by adding excess of test chemical in 14 L of Lake Superior water
- Controls: Dilution water
- Chemical name of vehicle: water
- Concentration of vehicle in test medium: Stock (650 µg/L liq-liq equil.)

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnows
- Source: ERL-Duluth, USA
- Age at study initiation: 30 days
- Length at study initiation: 20.9 mm (SD=2.2)
- Weight at study initiation: 128 mg (SD=35)

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

45.2 mg CaCO3/L (SD=0.3)

Test temperature:

25.2ºC (SD=0.2)

pH:

7.7 (SD=0.2)

Dissolved oxygen:

6.7 mg O2/L (SD=0.7)

Salinity:

42.2 mg CaCO3/L (SD=0.9)

Nominal and measured concentrations:

Nominal concentrations: 0, 130, 260, 390, 520 and 650 μg/L
Average measured concentrations: <31, 58, 140, 229, 303 and 424 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Six crystallizing dishes were used as test chambers in the electronic diluter with tank volumes of 200 mL; test solution depth: 4-5 cm
- Type (delete if not applicable): Closed
- Type of flow-through: Electronic diluter
- Renewal rate of test solution (frequency/flow rate): Flow rates of 21 mL/min to each tank produced toxicant concentrations of 58-424 μg/L
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): One
- No. of vessels per vehicle control (replicates): One
- Biomass loading rate: 6.4 g/L

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: Lake Superior water
- Preparation of dilution water: Water was passed through sand filters, ultraviolet sterilizers and 55 µm fibre filter. Test waters were then aerated and heated at 25 ± 1 °C
- Intervals of water quality measurement: Tank temperatures (ºC), dissolved oxygen (mg/L) and pH were measured daily in each of the test chambers where test organisms were still alive

OTHER TEST CONDITIONS
- Photoperiod: 16 hour/day light cycle; cool white fluorescent light
- Light intensity: 12-28 lumens

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Behavior and morphological aberrations were observed at 0, 24, 48, 72 and 96 hours

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

280 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL: 259-303 μg/L

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

179 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: Affected fish swam near the tank bottom, were hypoactive and underreactive to external stimuli, exhibited hemorrhaging and lost equilibrium prior to death.
- Mortality (96 hour) at <31, 58, 140, 229, 303 and 424 μg/L (measured) were 0, 0, 0, 0, 80 and 100%, respectively.
- Effect (96 hour) at <31, 58, 140, 229, 303 and 424 μg/L μg/L (measured) were observed in 0/10, 0/10, 0/10, 10/10, 10/10 and 10/10 fish respectively.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Measured concentrations were lower than nominal concentrations due to volatility.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

- LC50 or EC50 values were calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber Method (Hamilton et al., 1977).
- Mean toxicant concentrations used in the calculations were corrected for analytical spike recoveries

Sublethal observations / clinical signs:

Table 1: Mortalities

 

Hour	Control (0 µg/L)	A (130 µg/L)	B (260 µg/L)	C (390 µg/L)	D (520 µg/L)	E (650 µg/L)
Initial	10	10	9	10	10	10
24	0	0	0	0	7	8
48	0	0	0	0	8	10
72	0	0	0	0	8	10
96	0	0	0	0	8	10

Table 2: Effects

Hour	Control (0 µg/L)	A (130 µg/L)	B (260 µg/L)	C (390 µg/L)	D (520 µg/L)	E (650 µg/L)
Initial	10	10	9	10	10	10
24	0	0	0	10	10	10
48	0	0	0	10	10	10
72	0	0	0	10	10	10
96	0	0	0	10	10	10

 

Validity criteria fulfilled:

yes

Remarks:

(mortality in control < 10%; flow-through test conditions; dissolved O2 concentration > 60% of air saturation value; results based on measured concentrations)

Conclusions:

The 96h-LC50 of d-alpha pinene to fathead minnow (Pimephales promelas) was calculated to be 280 (259 -303) μg/L.

Executive summary:

In an acute aquatic toxicity study performed similarly to OECD guideline 203, groups (10/dose) of fathead minnows (Pimephales promelas) were exposed to d-alpha pinene at concentrations of <31, 58, 140, 229, 303 and 424 μg/L (measured) for 96 hours under flow-through conditions. Mortality, behaviour and morphological aberrations were observed at 24, 48, 72 and 96 hours. All validity criteria were fulfilled.Mortalities (96 hour) at <31, 58, 140, 229, 303 and 424 μg/L (measured) were 0, 0, 0, 0, 80 and 100%, respectively. Treatment-related effects (96 hour) at <31, 58, 140, 229, 303 and 424 μg/L (measured) were observed in 0/10, 0/10, 0/10, 10/10, 10/10 and 10/10 fish respectively. The 96 hour LC50, EC50 and NOEC of d-alpha pinene to fathead minnow (Pimephales promelas) were calculated to be 280 (259 -303) μg/L, 179 μg/L and 140 μg/L, respectively.

 

 

 

Reference 5

Endpoint:

short-term toxicity to fish

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

Remarks:

Study conducted similarly to OECD guideline 203 but without GLP compliance and with minor deviations (no data on acclimation period). The report is sufficiently well documented.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

No data on acclimation period

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 372, 744, 1120, 1490 and 1860 μg/L
- Sampling time: 0, 24, 48, 72 and 96 hours

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions was provided as liquid/liquid saturation units by adding excess of test chemical in 14 L of Lake Superior water
- Controls: Dilution water
- Chemical name of vehicle: water
- Concentration of vehicle in test medium: Stock (1810 µg/L liq-liq equil.)

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnows
- Source: ERL-Duluth, USA
- Age at study initiation: 32/34 days
- Length at study initiation: 21.8 ± 3.9 mm
- Weight at study initiation: 177 ± 84 mg

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

46.2 mg CaCO3/L (SD=0.3)

Test temperature:

24.4ºC (SD=0.6)

pH:

7.6 (SD=0.10)

Dissolved oxygen:

5.8 mg O2/L (SD=0.5)

Salinity:

39.5 mg CaCO3/L (SD=0.7)

Nominal and measured concentrations:

Nominal concentrations: 0, 372, 744, 1120, 1490 and 1860 μg/L
Average measured concentrations: <150, 178, 386, 595, 928 and 1100 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Six crystallizing dishes were used as test chambers in the electronic diluter with tank volumes of 200 mL; test solution depth: 4-5 cm
- Type (delete if not applicable): Closed
- Type of flow-through: Electronic diluter
- Renewal rate of test solution (frequency/flow rate): Flow rates of 21 mL/min to each tank produced toxicant concentrations of 178-1100 μg/L
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): One
- No. of vessels per vehicle control (replicates): One
- Biomass loading rate: 7.08 g/L

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: Lake Superior water
- Preparation of dilution water: Water was passed through sand filters, ultraviolet sterilizers and 55 µm fibre filter. Test waters were then aerated and heated at 25 ± 1ºC
- Intervals of water quality measurement: Tank temperatures (ºC), dissolved oxygen (mg/L) and pH were measured daily in each of the test chambers where test organisms were still alive

OTHER TEST CONDITIONS
- Photoperiod: 16 hour/day light cycle; cool white fluorescent light
- Light intensity: 12-28 lumens

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Behavior and morphological aberrations were observed at 0, 24, 48, 72 and 96 hours

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

702 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL: 619-796 μg/L

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

702 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CL: 619-796 μg/L

Details on results:

- Behavioural abnormalities: Affected fish were hyperactive and swam in a sporadic manner. They were overreactive to external stimuli and experienced hemorrhaging. Equilibrium loss was observed prior to death.
- Mortality (96 hour) at <150, 178, 386, 595, 928 and 1100 μg/L (measured) were 0, 0, 0, 20, 90 and 100%, respectively.
- Effect (96 hour) at <150, 178, 386, 595, 928 and 1100 μg/L (measured) were observed in 0/10, 0/10, 0/10, 2/10, 9/10 and 10/10 fish respectively.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Measured concentrations were lower than nominal concentrations due to volatility.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

- LC50 or EC50 values were calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber Method (Hamilton et al., 1977).
- Mean toxicant concentrations used in the calculations were corrected for analytical spike recoveries

Sublethal observations / clinical signs:

Table 1: Mortalities

 

Hour	Control (0 µg/L)	A (372 µg/L)	B (744 µg/L)	C (1120 µg/L)	D (1490 µg/L)	E (1860 µg/L)
Initial	10	10	10	10	10	10
24	0	0	0	2	9	6
48	0	0	0	2	9	8
72	0	0	0	2	9	10
96	0	0	0	2	9	10

Table 2: Effects

Hour	Control (0 µg/L)	A (372 µg/L)	B (744 µg/L)	C (1120 µg/L)	D (1490 µg/L)	E (1860 µg/L)
Initial	10	10	10	10	10	10
24	0	0	0	10	10	10
48	0	0	0	2	10	10
72	0	0	0	2	10	10
96	0	0	0	2	9	10

 

Validity criteria fulfilled:

yes

Remarks:

(mortality in control < 10%; flow-through test conditions; dissolved O2 concentration > 60% of air saturation value; results based on measured concentrations)

Conclusions:

The 96h-LC50 of d-limonene to fathead minnow (Pimephales promelas) was calculated to be 702 (619 -796) μg/L.

Executive summary:

In an acute aquatic toxicity study performed similarly to OECD guideline 203, groups (10/dose) of fathead minnows (Pimephales promelas) were exposed to d-limonene (purchased from Aldrich) at concentrations of <150, 178, 386, 595, 928 and 1100 μg/L (measured) for 96 hours under flow-through conditions. Mortality, behaviour and morphological aberrations were observed at 24, 48, 72 and 96 hours. All validity criteria were fulfilled. Mortalities (96 hour) at <150, 178, 386, 595, 928 and 1100 μg/L (measured) were 0, 0, 0, 20, 90 and 100%, respectively. Treatment-related effects (96 hour) at <150, 178, 386, 595, 928 and 1100 μg/L (measured) were observed in 0/10, 0/10, 0/10, 2/10, 9/10 and 10/10 fish respectively. The 96 hour LC50, EC50 and NOEC of d-limonene to fathead minnow (Pimephales promelas) were calculated to be 702 (619 -796) μg/L, 702 (619 -796) μg/L and 386 μg/L, respectively.

 

 

 

Reference 6

Endpoint:

short-term toxicity to fish

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

Remarks:

Study conducted similarly to OECD guideline 203 but without GLP compliance and with minor deviations (no data on acclimation period). The report is sufficiently well documented.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

No data on acclimation period

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 550, 1090, 1640, 2180 and 2730 μg/L
- Sampling time: 0, 24, 48, 72 and 96 hours

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions was provided as liquid/liquid saturation units by adding excess of test chemical in 14 L of Lake Superior water
- Controls: Dilution water
- Chemical name of vehicle: water
- Concentration of vehicle in test medium: Stock (2730 µg/L liq-liq equil.)

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnows
- Source: ERL-Duluth, USA
- Age at study initiation: 34 days
- Length at study initiation: 19.1 mm (SD=2.1)
- Weight at study initiation: 85 mg (SD=34)

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

no data

Test temperature:

25.4 ºC (SD=0.3)

pH:

7.4 (SD=0.1)

Dissolved oxygen:

6.9 mg O2/L (SD=0.3)

Nominal and measured concentrations:

Nominal concentrations: 0, 550, 1090, 1640, 2180 and 2730 μg/L
Average measured concentrations: <49, 251, 564, 961, 1380 and 1890 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Six crystallizing dishes were used as test chambers in the electronic diluter with tank volumes of 200 mL; test solution depth: 4-5 cm
- Type (delete if not applicable): Closed
- Type of flow-through: Electronic diluter
- Renewal rate of test solution (frequency/flow rate): Flow rates of 21 mL/min to each tank produced toxicant concentrations of 251-1890 μg/L
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): One
- No. of vessels per vehicle control (replicates): One
- Biomass loading rate: 4.25 g/L

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: Lake Superior water
- Preparation of dilution water: Water was passed through sand filters, ultraviolet sterilizers and 55 µm fibre filter. Test waters were then aerated and heated at 25 ± 1ºC
- Intervals of water quality measurement: Tank temperatures (ºC), dissolved oxygen (mg/L) and pH were measured daily in each of the test chambers where test organisms were still alive

OTHER TEST CONDITIONS
- Photoperiod: 16 hour/day light cycle; cool white fluorescent light
- Light intensity: 12-28 lumens

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Behavior and morphological aberrations were observed at 0, 24, 48, 72 and 96 hours

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

720 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL: 618-839 μg/L

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

688 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CL: 606-782 μg/L

Details on results:

- Behavioural abnormalities: Affected fish were hypoactive and underreactive to external stimuli. They were also darkly colored and lost equilibrium prior to death.
- Mortality (96 hour) at <49, 251, 564, 961, 1380 and 1890 μg/L (measured) were 0, 0, 10, 90, 100 and 100%, respectively.
- Effect (96 hour) at <49, 251, 564, 961, 1380 and 1890 μg/L (measured) were observed in 0/10, 0/10, 1/10, 10/10, 10/10 and 10/10 fish respectively.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Measured concentrations were lower than nominal concentrations due to volatility.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

- LC50 or EC50 values were calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber Method (Hamilton et al., 1977).
- Mean toxicant concentrations used in the calculations were corrected for analytical spike recoveries

Sublethal observations / clinical signs:

Table 1: Mortalities

 

Hour	Control (0 µg/L)	A (550 µg/L)	B (1090 µg/L)	C (1640 µg/L)	D (2180 µg/L)	E (2730 µg/L)
Initial	10	10	10	10	10	10
24	0	0	1	9	10	10
48	0	0	1	9	10	10
72	0	0	1	9	10	10
96	0	0	1	9	10	10

Table 2: Effects

Hour	Control (0 µg/L)	A (550 µg/L)	B (1090 µg/L)	C (1640 µg/L)	D (2180 µg/L)	E (2730 µg/L)
Initial	10	10	10	10	10	10
24	0	0	1	10	10	10
48	0	0	10	10	10	10
72	0	0	10	10	10	10
96	0	0	1	10	10	10

 

Validity criteria fulfilled:

yes

Remarks:

(mortality in control < 10%; flow-through test conditions; dissolved O2 concentration > 60% of air saturation value; results based on measured concentrations)

Conclusions:

The 96h-LC50 of d-limonene to fathead minnow (Pimephales promelas) was calculated to be 720 (618 -839) μg/L.

Executive summary:

In an acute aquatic toxicity study performed similarly to OECD guideline 203, groups (10/dose) of fathead minnows (Pimephales promelas) were exposed to d-limonene (purchased from Fluorostat) at concentrations of <49, 251, 564, 961, 1380 and 1890 μg/L (measured) for 96 hours under flow-through conditions. Mortality, behaviour and morphological aberrations were observed at 24, 48, 72 and 96 hours. All validity criteria were fulfilled. Mortalities (96 hour) at <49, 251, 564, 961, 1380 and 1890 μg/L (measured) were 0, 0, 10, 90, 100 and 100%, respectively. Treatment-related effects (96 hour) at <49, 251, 564, 961, 1380 and 1890 μg/L (measured) were observed in 0/10, 0/10, 1/10, 10/10, 10/10 and 10/10 fish respectively. The 96 hour LC50, EC50 and NOEC of d-limonene to fathead minnow (Pimephales promelas) were calculated to be 720 (618 -839) μg/L, 688 (606 -782) μg/L and 251 μg/L, respectively.

 

 

 

Reference 7

Endpoint:

short-term toxicity to fish

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

Remarks:

Study conducted similarly to OECD guideline 203 but without GLP compliance and with minor deviations (no data on acclimation period). The report is sufficiently well documented.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

No data on acclimation period

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 450, 900, 1340, 1790 and 2240 μg/L
- Sampling time: 0, 24, 48, 72 and 96 hours

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions was provided as liquid/liquid saturation units by adding excess of test chemical in 14 L of Lake Superior water
- Controls: Dilution water
- Chemical name of vehicle: water
- Concentration of vehicle in test medium: Stock (2240 µg/L liq-liq equil.)

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnows
- Source: ERL-Duluth, USA
- Age at study initiation: 30 days
- Length at study initiation: 16.3 mm (SD=1.6)
- Weight at study initiation: 59 mg (SD=18)

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

45.1 mg CaCO3/L (SD=0.2)

Test temperature:

25.0ºC (SD=0.2)

pH:

7.6 (SD=0.12)

Dissolved oxygen:

6.3 mg O2/L (SD=0.9)

Salinity:

42.6 mg CaCO3/L (SD=0.7)

Nominal and measured concentrations:

Nominal concentrations: 0, 450, 900, 1340, 1790 and 2240 μg/L
Average measured concentrations: <33, 279, 645, 902, 1360 and 1670 μg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Six crystallizing dishes were used as test chambers in the electronic diluter with tank volumes of 200 mL; test solution depth: 4-5 cm
- Type (delete if not applicable): Closed
- Type of flow-through: Electronic diluter
- Renewal rate of test solution (frequency/flow rate): Flow rates of 21 mL/min to each tank produced toxicant concentrations of 279-1670 μg/L
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): One
- No. of vessels per vehicle control (replicates): One
- Biomass loading rate: 2.66 g/L

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: Lake Superior water
- Preparation of dilution water: Water was passed through sand filters, ultraviolet sterilizers and 55 µm fibre filter. Test waters were then aerated and heated at 25 ± 1 °C
- Intervals of water quality measurement: Tank temperatures (ºC), dissolved oxygen (mg/L) and pH were measured daily in each of the test chambers where test organisms were still alive

OTHER TEST CONDITIONS
- Photoperiod: 16 hour/day light cycle; cool white fluorescent light
- Light intensity: 12-28 lumens

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Behavior and morphological aberrations were observed at 0, 24, 48, 72 and 96 hours

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1 210 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL: 1110-1330 μg/L

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

763 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: Affected fish swam in an almost vertical position, were underreactive to external stimuli and had convulsions. Equilibrium loss was observed prior to death.
- Mortality (96 hour) at <33, 279, 645, 902, 1360 and 1670 μg/L (measured) were 0, 0, 0, 0, 70 and 100%, respectively.
- Effect (96 hour) at <33, 279, 645, 902, 1360 and 1670 μg/L (measured) were observed in 0/10, 0/10, 0/10, 10/10, 10/10 and 10/10 fish respectively.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Measured concentrations were lower than nominal concentrations due to volatility.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

- LC50 or EC50 values were calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber Method (Hamilton et al., 1977).
- Mean toxicant concentrations used in the calculations were corrected for analytical spike recoveries

Sublethal observations / clinical signs:

Table 1: Mortalities

 

Hour	Control (0 µg/L)	A (450 µg/L)	B (900 µg/L)	C (1340 µg/L)	D (1790 µg/L)	E (2240 µg/L)
Initial	9	10	10	10	10	10
24	0	0	0	0	5	9
48	0	0	0	0	6	10
72	0	0	0	0	7	10
96	0	0	0	0	7	10

Table 2: Effects

Hour	Control (0 µg/L)	A (450 µg/L)	B (900 µg/L)	C (1340 µg/L)	D (1790 µg/L)	E (2240 µg/L)
Initial	9	10	10	10	10	10
24	0	0	0	0	7	10
48	0	0	0	10	10	10
72	0	0	0	0	10	10
96	0	0	0	10	10	10

 

Validity criteria fulfilled:

yes

Remarks:

(mortality in control < 10%; flow-through test conditions; dissolved O2 concentration > 60% of air saturation value; results based on measured concentrations)

Conclusions:

The 96h-LC50 of terpinolene to fathead minnow (Pimephales promelas) was calculated to be 1210 (1110 -1330) μg/L.

Executive summary:

In an acute aquatic toxicity study performed similarly to OECD guideline 203, groups (10/dose) of fathead minnows (Pimephales promelas) were exposed to terpinolene at concentrations of <33, 279, 645, 902, 1360 and 1670 μg/L (measured) for 96 hours under flow-through conditions. Mortality, behaviour and morphological aberrations were observed at 24, 48, 72 and 96 hours. All validity criteria were fulfilled.Mortalities (96 hour) at <33, 279, 645, 902, 1360 and 1670 μg/L (measured) were 0, 0, 0, 0, 70 and 100%, respectively. Treatment-related effects (96 hour) at <33, 279, 645, 902, 1360 and 1670 μg/L (measured) were observed in 0/10, 0/10, 0/10, 10/10, 10/10 and 10/10 fish respectively. The 96 hour LC50, EC50 and NOEC of terpinolene to fathead minnow (Pimephales promelas) were calculated to be 1210 (1110 -1330)μg/L, 763 μg/L and 645 μg/L, respectively.

 

 

 

Reference 8

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

Test method according to US EPA (1975): "Methods for acute toxicity tests with fish, macroinvertebrates and amphibiens".

Qualifier:

according to guideline

Guideline:

other: US EPA (1975): "Methods for acute toxicity tests with fish, macroinvertebrates and amphibiens"

Deviations:

not specified

GLP compliance:

no

Analytical monitoring:

not specified

Vehicle:

not specified

Test organisms (species):

Cyprinodon variegatus

Details on test organisms:

TEST ORGANISM
- Common name: Sheepshead Minnows
- Source: Animals were hatched and reared at BMRL or obtained from the EPA's Environmental Research Laboratory, Gulf Breeze, FL.
- Age at study initiation (mean and range, SD): 14-28 days old posthatch
- Length at study initiation (length definition, mean, range and SD): 8 - 15 mm standard length
- Weight at study initiation (mean and range, SD):
- Method of breeding:
- Maintenance of the brood fish:

ACCLIMATION
- Acclimation conditions (same as test or not): Flowing, filtered (10-µm) seawater
- Type and amount of food/Feeding frequency: Fish were fed 24-h Artemia salina nauplii (San Francisco Bay Brand) daily until they were used as test animals.
- Health during acclimation (any mortality observed): Fish mortality during the 48-h period immediately before each test was less than 3% and all fish tested appeared to be in good condition.

FEEDING DURING TEST: No

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

25-31 ºC

Salinity:

1.0-3.1 %

Nominal and measured concentrations:

No data on test concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: Tests were conducted in either 4-L glass jars that contained 3 L of test solution or 19-L glass jars that contained 15 L
- Aeration: No
- No. of organisms per vessel: 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: All dilution water was filtered (5-µm), natural seawater of ambient salinity.
- Intervals of water quality measurement: The dissolved oxygen concentration was measured in each test container at initiation of testing and daily thereafter; pH was measured in the control and the low and high test concentrations at the initiation and after 96 h of testing.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortality was recorded at 24, 48, 72, and 96 h.

Reference substance (positive control):

not specified

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (95% confidence limits: 1.6-2.2 ppm)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (95% confidence limits: 1.6-2.4 ppm)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (95% confidence limits: 1.6-2.4 ppm)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (95% confidence limits: 1.0-3.2 ppm)

Reported statistics and error estimates:

Based on the results of the tests, 24-, 48-, 72-, and 96-h LC50's and their 95% confidence limits were calculated when possible. Calculations were performed with a Hewlett Packard Model 9815A calculator programmed (STEPHAN 1977 and 1978 personal communication) to scan the data base and to select the most appropriate of three statistical methods in the following order: moving average angle analysis, probit analysis, or binomial probability. The method selected was determined by the characteristics of the data, that is, the presence or absence of 0% and 100% mortality and the number of concentrations in which >0%<100% mortality occurred. The computer scanned the data, identified the most suited method, and performed the analyses. No correction was made for control mortality.

Sublethal observations / clinical signs:

The LC50 (96 h) was 1.9 ppm (basis for effect: mortality). The NOEC was 1.0 ppm.

Validity criteria fulfilled:

not specified

Remarks:

(No data on control results. There is no evidence that test substance concentration had been maintained throughout the test period).

Conclusions:

The LC50 (96 h) was 1.9 ppm (basis for effect: mortality).

Executive summary:

A marine toxicity static test were conducted at EG&G Bionomics Marine Research Laboratory (BMRL) with sheepshead minnows (Cyprinodon variegatus). Test method was according to US EPA (1975): "Methods for acute toxicity tests with fish, macroinvertebrates and amphibiens". Camphene was tested for 96 hours at 25 -31 ºC. Mortality was recorded at 24, 48, 72, and 96 h. The 96 h-LC50 was 1.9 ppm (basis for effect: mortality). The NOEC was 1.0 ppm.

Reference 9

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

22 April 1988 - 26 April 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Test method was similar to guideline OECD 203

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
- Sample storage conditions before analysis: All samples were analyzed immediately or adequately preserved for later analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A diluted stock of the test substance (665 mg/L) was obtained in a 18.9 L stainless steel carbonation vessel. Lake Superior water plus the test substance were added to the vessel and blended vigorously.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnows
- Source: EPA Environmental Research Laboratory-Duluth and the University of Wisconsin-Superior campus
- Age at study initiation: 28 days old
- Length at study initiation (length definition, mean, range and SD): 19.1 mm (mean), 0.933 mm (SD)
- Weight at study initiation (mean and range, SD): 0.097 g (mean), 0.0126 g (SD)
- Method of breeding: Culture chambers were provided with asbestos or plastic pipes (cut in half longitudinally) as spawning substrates, to which the naturally spawned and fertilized embryos became attached to the undersides. The substrates, with intact embryos, were then transferred into another 25ºC bath where hatching ocurred or substrates were removed just prior to hatching at the UW-Superior culture unit, then placed in a rearing bath. Larvae were fed 40 to 48 hr old brine shrimp nauplii (Bio-Marine Research, Inc., Hawthorne, CA or San Francisco Bay Brand, Inc., Newarkk, CA) in excess two times daily (once daily on weekens).


ACCLIMATION
Fish that were 27-44 days old were used in the toxicity tests.
- Health during acclimation (any mortality observed): Only groups of fish having healthy appearance and no history of unusual thermal exposure or abnormally high mortality rate were used for tests.

FEEDING DURING TEST
Test fish were not fed 24 hr before or during a test.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

43.9 (SD=0.25) mg/L (as CaCO3)

Test temperature:

25.1 (SD=0.62) ºC

pH:

7.9 (SD=0.06)

Dissolved oxygen:

6.9 (SD=0.26) mg/L

Nominal and measured concentrations:

Nominal concentrations: 0, 48.2, 74.1, 114, 176 and 270 mg/L
Measured concentrations (corrected aveg.): <0.99, 40.3, 50.8, 75.9, 124 and 203 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 1 L tank
- Type of flow-through (e.g. peristaltic or proportional diluter): continuous-flow modified mini-diluter (Benoit et al.,1982)
- Renewal rate of test solution (frequency/flow rate): 14.4 volumes per day (10 mL/min)
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 1.940 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Lake Superior water filtered through sand and a cotton fiber filter.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Number of dead fish was noted approximately every 24 hr after the beginning of a test, at which time they were also removed. The following observations of fish behaviour and toxic signs were made at 2-8, 24, 48, 72 and 96 hr: Loss of equilibria, general activity, locomotor activities, schooling behaviour, location in tank, startle response, body movements, body pigmentation and body of live fish.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.5

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

102 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: LC50 calculated using corrected average of measured concentrations and trimmed spearman-karber method with 95% conf. lim. = 95.4-109 mg/L

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

102 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: EC50 calculated using corrected average of measured concentrations and trimmed spearman-karber method with 95% conf. lim. = 95.4-109 mg/L

Details on results:

- Behavioural abnormalities: Affected fish lost schooling behaviour, swam near the tank surface and were hyperactive. They were also overreactive to external stimuli, had increased respiaration and lost equilibrium prior to death.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: samples were not taken at 96 h. Measured concentrations were much lower than nominal concentrations due to volatility.

Reported statistics and error estimates:

LC50 and EC50 with corresponding 95% confidence intervals were calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber Method (Hamilton et al.,1977)

Sublethal observations / clinical signs:

Table1.Mortalities

Time (h)	Nominal concentrations (mg/L)
	0	48.2	74.1	114	176	270
Initial	20	20	20	20	20	20
5	-	-	-	-	8	20
24	-	-	-	-	18	20
48	-	-	-	-	18	20
72	-	-	-	-	18	20
96	-	-	-	-	18	20

 

Table 2. Effect

Time (h)	Nominal concentrations (mg/L)
	0	48.2	74.1	114	176	270
Initial	20	20	20	20	20	20
5	-	-	-	-	8	20
24	-	-	-	-	18	20
48	-	-	-	-	18	20
72	-	-	-	-	18	20
96	-	-	-	-	18	20

Validity criteria fulfilled:

yes

Remarks:

(mortality in control was 0%; constant conditions were maintained; the dissolved oxygen concentration was within the acceptable range; the concentrations were maintained during the tests).

Conclusions:

The LC50 of test item in Fathead Minnows (Pimephales promelas) at 96 h exposure period was determined to be 102 mg/L.

Executive summary:

An acute toxicity test in Fathead Minnows (Pimephales promelas) was performed following a method similar to OECD guideline 203. 20 fish per group were exposed over an exposure period of 96 h in a flow-through system to the following nominal concentrations: 0 (control), 48.2, 74.1, 114, 176 and 270 mg/L. Gas-liquid chromatography was used to monitor the concentration throughout the test. LC50 and EC50 with corresponding 95% confidence intervals were calculated using corrected average of measured concentrations and trimmed spearman-karber method. Both 96h-EC50 and 96h-LC50 were determined to be 102 mg/L (95% CL = 95.4-109 mg/L).

Reference 10

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Test method according to guideline OECD 203

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: analyses of the test substance were performed at beginning of exposure and after 48 h.

Vehicle:

no

Test organisms (species):

Oryzias latipes

Details on test organisms:

TEST ORGANISM
- Common name: Ricefish

FEEDING DURING TEST
Test fish were not fed during test.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

24 ± 2ºC

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 1.0, 1.8, 3.2, 5.6 and 10 mg/L
Measured concentrations: Since the measured concentrations of the test substance exceeded the nominal concentrations by ± 20%, the results were calculated from the measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 9 L tank
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): running water type (using continuous dilution apparatus using metering pump)
- Renewal rate of test solution (frequency/flow rate): ca. 8 volumes per day (50 mL/min)
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate:

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light / 8 hours dark

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

2 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

3.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Reported statistics and error estimates:

LC50 was calculated by binomial test.

Validity criteria fulfilled:

not specified

Conclusions:

The LC50 of test item in Ricefish (Oryzias latipes) at 96 h exposure period was determined to be 2 mg/L.

Executive summary:

An acute toxicity test in Ricefish (Oryzias latipes) was performed following a method similar to OECD guideline 203. 10 fish per group were exposed over an exposure period of 96 h in a flow-through system to the following nominal concentrations: 0 (control), 1.0, 1.8, 3.2, 5.6 and 10mg/L. Gas chromatography mass spectrometry was used to monitor the concentration throughout the test. The results were calculated from the measured concentrations. The 96h-LC50 was determined to be 2 mg/L. Also 96h-LC0 and 96h-LC100 were calculated to be 1.2 and 3.4 mg/L respectively.

Description of key information

Key study. Test method according to OECD 203, GLP study. The 96h-LL50 of the test substance to Cyprinus carpio (common carp) was found to be 34.67 mg/L (95% confidence limits: 30.05- 40.1 mg/L).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

34.67 mg/L

Additional information

Supporting studies with individual components: Data on short-term toxicity to fish of the main constituents are available from experimental data:

Alpha terpineol: Test method according to Environment Canada (Stroh J, 1998). The 96h-LC50 was determined to be 6.6 mg/L in rainbow trout (Oncorhynchus mykiss).

Terpinolene: Test method similar to OECD TG 203 (Broderius S, 1990). The 96h-LC50 was determined to be 1.21 mg/L in fathead minnow (Pimephales promelas).

Cineole: Test method similar to OECD TG 203 (Geiger D L, 1988). The 96h-LC50 was determined to be 102 mg/L in fathead minnow (Pimephales promelas).

D-Limonene (test001): Test method similar to OECD TG 203 (Broderius S, 1990). The 96h-LC50 was determined to be 0.702 mg/L in fathead minnow (Pimephales promelas).

D-Limonene(test002): Test method similar to OECD TG 203 (Broderius S, 1990). The 96h-LC50 was determined to be 0.720 mg/L in fathead minnow (Pimephales promelas).

Camphene: Test method according to US EPA (1975): "Methods for acute toxicity tests with fish, macroinvertebrates and amphibiens". The 96 h-LC50 was determined to be 1.9 ppm.

Alpha terpinene: Test method similar to OECD TG 203 (Broderius S, 1990). The 96h-LC50 was determined to be 3.15 mg/L in fathead minnow (Pimephales promelas).

D-alpha pinene: Test method similar to OECD TG 203 (Broderius S, 1990). The 96h-LC50 was determined to be 0.28 mg/L in fathead minnow (Pimephales promelas).

p-cymene: Test method similar to OECD TG 203 (MITI, 1998). The 96h-LC50 was determined to be 2 mg/L in Ricefish (Oryzias latipes).