1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-nortall-oil alkyl derivs.

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:

The study was conducted between 01 December 2006 and 10 July 2007.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.010, 0.022, 0.046, 0.10, 0.22, and 0.46 mg/L.
- Sampling method: Small volumes of the test media and the control (1.0 mL) were taken from all test flasks after 24, 48 and 72 hours of exposure, and were not replaced. In addition, after 72 hours of exposure, a sample was taken from the control and from a test concentration with reduced algal growth (nominal 0.046 mg/L).
- Sample storage conditions before analysis: All samples were stored deep-frozen (at about -20 °C) and protected from light immediately after sampling. Based on preexperiments (without GLP) for investigation of the storage stability, the test item is sufficiently stable in the test water under these storage conditions.

Vehicle:

not specified

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The following nominal concentrations of 1H-Imidazole-1-ethanol, 4,5-dihydro, 2-nortall-oil, alky derivatives were tested: 0.010, 0.022, 0.046, 0.10, 0.22, and 0.46 mg/L. A stock solution of nominal 46 mg/L was prepared by dissolving 45.9 mg of the test item completely in 1000 mL of test water using ultrasonic treatment (15 minutes) and intense stirring (15 minutes at room temperature). This intensively mixed stock solution was used in a series of dilutions to prepare the test media of all test item concentrations. The test media were prepared just before the start of the test (= addition of algae). The actual concentrations of the test item in the test media were analytically determined.
- Differential loading: not applicable
- Controls: A control was tested in parallel (test water without test item).
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No remarkable observations, clear test medium.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum)
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, D-37073 Göttingen, Germany)
- Age of inoculum (at test initiation): Inoculum was taken from an exponentially growing pre-culture, which was set up four days prior to the test under the same conditions as in the test. One day before the start of the test, the preculture was diluted threefold to keep the algae in exponential growth.
- Method of cultivation: Volumes of 15 mL algal suspension for each replicate were continuously stirred by magnetic stirrers in 50-mL Erlenmeyer flasks. The test design included three replicates per test concentration and six replicates of the control.

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions: the same as test conditions
- Any deformed or abnormal cells observed: no abnormalities observed

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines. Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L as CaCO3).

Test temperature:

The test was performed in a temperature controlled water bath at a temperature of 22-24°C.

pH:

At the start of the test, the pH values in the test media and the control ranged from 8.0 to 8.3. At the end of the test, pH values of 8.1 to 9.1 were measured.

Dissolved oxygen:

Not measured.

Salinity:

Not applicable.

Nominal and measured concentrations:

At the start of the test, the measured test item concentrations in the analyzed test media ranged from 75 to 100% of the nominal values.
Nominal concentrations at the start of the test: 0.010, 0.022, 0.046, 0.10, 0.22, and 0.46 mg/L.
Measured concentrations at the start of the test: not analyzed, 0.022, 0.040, 0.075, 0.19, 0.45 mg/L.
At the end of the test, 84 to 98% of the initial measured values were found. Thus, the test item was sufficiently stable during the test period and the reported biological results are based on the initial measured test item concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Volumes of 15 mL algal suspension for each replicate were continuously stirred by magnetic stirrers in 50-mL Erlenmeyer flasks. The flasks were covered with glass dishes.
- Renewal rate of test solution: no
- Initial cells density: The test was started (0 hours) by inoculation of 10,000 algal cells per mL of test medium. These cells were taken from an exponentially growing pre-culture, which was set up four days prior to the test under the same conditions as in the test.
- Control end cells density: The algal cell densities in the samples were determined after 24, 48 and 72 hours by counting with an electronic particle counter (Coulter Counter®, Model ZM), with at least two measurements per sample.
- No. of vessels per concentration (replicates): three replicates per test concentration
- No. of vessels per control (replicates): six replicates


GROWTH MEDIUM
- Standard medium used: The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines.
- Detailed composition if non-standard medium was used:
Analytical grade salts were dissolved in sterile purified water to obtain the following final nominal concentrations:
Macro-nutrients:
NaHCO3 50.0 mg/L
CaCl2 × 2 H2O 18.0 mg/L
NH4Cl 15.0 mg/L
MgSO4 × 7 H2O 15.0 mg/L
MgCl2 × 6 H2O 12.0 mg/L
KH2PO4 1.6 mg/L
Trace elements:
Na2EDTA × 2 H2O 100.0 μg/L
FeCl3 × 6 H2O 80.0 μg/L
MnCl2 × 4 H2O 415.0 μg/L
H3BO3 185.0 μg/L
Na2MoO4 × 2 H2O 7.0 μg/L
ZnCl2 3.0 μg/L
CoCl2 × 6 H2O 1.5 μg/L
CuCl2 × 2 H2O 0.01 μg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines.
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH was measured and recorded in each test concentration and the control at the start and at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was also recorded daily.

OTHER TEST CONDITIONS
- Photoperiod and light intensity and quality: The flasks were continuously illuminated at a measured light intensity of about 8300 Lux (mean value), range: 7960 to 8790 Lux (minimum and maximum value of measurements at nine places distributed over the experimental area at the surface of the test media). This illumination was achieved by fluorescent tubes (Philips TLD 36W/840), installed about 35 cm above the test flasks.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Small volumes of the test media and the control (1.0 mL) were taken from all test flasks after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities in the samples were determined by counting with an electronic particle counter (Coulter Counter®, Model ZM), with at least two measurements per sample.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0 - 2.2
- Test concentrations: The following nominal concentrations were used in the test: 0.010, 0.022, 0.046, 0.10, 0.22, and 0.46 mg/L.
- Results used to determine the conditions for the definitive study: The test concentrations were based on the results of a range-finding test (without GLP).

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.084 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.073-0.098

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.022 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.049 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 0.046-0.051

Details on results:

The influence of the test item 1H-Imidazole-1-ethanol, 4,5-dihydro, 2-nortall-oil, alky derivatives on the growth of Pseudokirchneriella subcapitata is shown in Table 1–Table 4 and Figure 1–Figure 3. The test item had a statistically significant inhibitory effect on the growth (growth rate and yield) of Pseudokirchneriella subcapitata after the test period of 72 hours at the initial measured test concentrations of 0.040 mg/L and above (results of Dunnett’s tests, one-sided, α = 0.05, Table 2 and Table 3). Thus, this test concentration was determined as the 72-hour LOEC (lowest concentration tested with toxic effects).
The 72-hour NOEC (highest concentration tested without toxic effects after a test period of 72 hours) was determined to be 0.022 mg/L, since up to and including this test concentration the mean growth rate and yield of the algae were not statistically significantly lower than in the control (Table 2 and 3).

In the control the cell density increased from a nominal N = 1 × 104 cells/mL at the start of the test (0 hours) to N = 98 × 104 cells/mL (mean value) after 72 hours (Table 1). The validity criterion of increase of cell density by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates (section-by-section growth rates, see Table 4) during 72 hours was 8.0% and thus lower than the maximum of 35% given by the OECD test guideline. The coefficient of variation of the average specific growth rates in the control replicates after 72 hours was 1.4% and, thus, below the maximum of 7% indicated by the OECD test guideline. Therefore, all validity criteria given by the test guidelines were fulfilled.

The microscopic examination of the algal cells after 72 hours exposure showed no difference between the algae growing at the test concentration of nominal 0.046 mg/L and the algal cells in the control. The shape and size of the algal cells growing in test media containing the test item at and up to this test concentration were not affected.

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

Results with reference substance (positive control):

Potassium dichromate is tested as a positive control at least once a year to demonstrate satisfactory conditions of the test.
The latest result of the positive control test in 2006 (72-h EC50 for the growth rate: 1.70 mg/L, RCC Study No. B13151) showed that the toxic performance was valid and within the historical range of the Harlan Laboratories (from 2000 to 2006: 72-h EC50: 0.71–1.74 mg/L).

Table 1: Algal cell density during the 72-hour test period

Nominal (initial measured) test item concentration	Flask no.	Density of algal cells (cell number x 10 000/mL) after
		24 h		48 h		72 h
control	1	4.4	4.9	22.8	23.6	88.0	85.4
	2	5.2	4.6	24.6	23.9	97.8	97.4
	3	4.9	4.5	24.2	24.2	95.8	97.2
	4	4.2	4.4	23.1	23.3	102.8	100.8
	5	5.3	4.8	23.8	23.9	100.8	99.4
	6	5.2	4.9	24.8	25.6	105.4	102.4
	m s m	4.8 0.3 6		24.0 0.8 6		97.8 6.1 6
0.010 (n.a)	1	4.8	4.3	25.0	23.8	98.6	100.6
	2	4.8	4.5	23.9	23.4	102.4	99.8
	3	5.2	4.8	25.2	23.6	103.4	102.0
	m s m	4.3 0.1 3		24.1 0.4 3		101.1 1.6 3
0.022 (0.022 mg/L)	1	4.2	4.1	23.7	22.9	100.4	98.6
	2	4.4	4.3	23.3	22.7	91.6	90.4
	3	4.5	4.0	22.5	22.2	96.4	99.6
	m s m	4.3 0.1 3		22.9 0.5 3		96.2 4.5 3
0.046 (0.040 mg/L)	1	3.5	3.7	19.8	20.4	81.2	83.8
	2	4.0	3.6	18.5	17.9	69.2	70.6
	3	3.6	3.4	17.2	16.9	64.4	63.2
	m s m	3.6 0.5 3		18.4 1.5 3		72.1 9.5 3
0.10 (0.075 mg/L)	1	3.3	3.4	4.9	4.7	7.6	7.0
	2	2.4	2.5	5.9	5.8	13.0	12.2
	3	2.8	2.5	5.9	5.8	6.6	6.4
	m s m	2.8 0.5 3		5.5 0.6 3		8.8 3.3 3
0.22 (0.19 mg/L)	1	2.5	2.6	2.7	2.3	2.6	2.2
	2	2.6	2.7	2.5	2.4	2.4	2.2
	3	2.3	2.3	2.1	2.1	3.2	2.4
	m s m	2.5 0.2 3		2.3 0.2 3		2.5 0.3 3
0.46 (0.45 mg/L)	1	1.3	1.2	1.5	1.0	1.0	0.6
	2	1.3	1.1	1.3	1.0	1.6	1.0
	3	1.4	1.6	1.4	1.0	1.0	0.8
	m s m	1.3 0.1 3		1.2 0.1 3		1.0 0.3 3

m: mean value; s: standard deviation; n: number of flasks

At the start, 10,000 algal cells/mL were inoculated.

n.a.: not analyzed

Table 2. Growth rates μ and percentage inhibition of μ (Ir) during the test period

Nominal test item concentration (mg/L)	Initial measured concentration (mg/L)	Growth rate μ and % inhibition of μ
		0-24 h		0-48 h		0-72 h
		μ (1/day)	Ir(%)	μ (1/day)	Ir(%)	μ (1/day)	Ir(%)
control	---	1.562	0.0	1.588	0.0	1.527	0.0
0.010	n.a.	1.554	0.5	1.592	-0.2	1.539	-0.8
0.022	0.022	1.447	7.4	1.565	1.5	1.522	0.3
0.046	0.040	1.290*	17.4	1.456*	8.3	1.424*	6.7
0.10	0.075	1.027*	34.3	0.850*	46.5	0.710*	53.5
0.22	0.19	0.915*	41.4	0.426*	73.2	0.304*	80.1
0.46	0.45	0.284*	81.8	0.091*	94.3	-0.007*	100.5

- % inhibition: increase in growth relative to that of control

*: mean value significantly lower than in control

(according to a Dunnett’s-test, one-sided smaller,α= 0.05)

n.a.: not analyzed

Table 3: Yield (y) and percentage inhibition of y (Iy) during the test period

Nominal test item concentration (mg/L)	Initial measured concentration (mg/L)	Yield y and % inhibition of y
		0-24 h		0-48 h		0-72 h
		yield	Iy(%)	yield	Iy(%)	yield	Iy(%)
control	---	3.775	0.0	22.983	0.0	96.767	0.0
0.010	n.a.	3.733	1.1	23.150	-0.7	100.133	-3.5
0.022	0.022	3.250*	13.9	21.883	4.8	95.167	1.7
0.046	0.040	2.633*	30.2	17.450*	24.1	71.067*	26.6
0.10	0.075	1.817*	51.9	4.500*	80.4	7.800*	91.9
0.22	0.19	1.500*	60.3	1.350*	94.1	1.500*	98.4
0.46	0.45	0.333*	91.2	0.200*	99.1	0.000*	100.0

y x 10,000

-% inhibition: increase in growth relative to that of control

*: mean value significantly lower than in control (according to a Dunnett’s-test, one-sided smaller,α= 0.05)

n.a.: not analyzed

Table 4: Section-by-section growth rates

Nominal test item concentration (mg/L)	Initial measured concentration (mg/L)	Section-by-section growth rates (1/day) and % inhibition of the growth rates
		0-24 h		0-48 h		0-72 h
		μ (1/day)	Ir(%)	μ (1/day)	Ir(%)	μ (1/day)	Ir(%)
control	---	1.562	0.0	1.615	0.0	1.404	0.0
0.010	n.a.	1.554	0.5	1.630	-0.9	1.432	-2.0
0.022	0.022	1.447	7.4	1.684	-4.2	1.435	-2.2
0.046	0.040	1.290	17.4	1.623	-0.5	1.359	3.2
0.10	0.075	1.027	34.3	0.674	58.3	0.431	69.6
0.22	0.19	0.915	41.4	-0.063	103.9	0.061	95.6
0.46	0.45	0.284	81.8	-0.102	106.3	-0.204	114.5

- % inhibition: increase in growth relative to that of control

n.a.: not analyzed

Validity criteria fulfilled:

yes

Conclusions:

The test item had a statistically significant inhibitory effect on the growth (growth rate and yield) of Pseudokirchneriella subcapitata after the test period of 72 hours at the initial measured test concentrations of 0.040 mg/L and above. Thus, this test concentration was determined as the 72-hour LOEC. The 72-hour NOEC was determined to be 0.022 mg/L, since up to and including this test concentration the mean growth rate and yield of the algae were not statistically
significantly lower than in the control.

Executive summary:

Introduction

The influence of the test item 1H-Imidazole-1-ethanol, 4,5-dihydro, 2-nortall-oil, alky derivatives on the growth of the freshwater green algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) was investigated in a 72-hour static test according to the EU Commission Directive 92/69/EEC, C.3 (1992), and the OECD Guideline 201 (2006).

Methods

Exponentially growing cultures of this unicellular algal species were exposed to various concentrations of the test item under defined conditions. A static, non-renewal exposure system was used. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours and, thus, over several algal generations. The nominal test concentrations were 0.010, 0.022, 0.046, 0.10, 0.22, and 0.46 mg/L in parallel to a control.

At the start of the test, the measured test item concentrations in the analyzed test media ranged from 75 to 100% of the nominal values. At the end of the test, 84 to 98% of the initial measured values were found. Thus, the test item was sufficiently stable during the test period and the reported biological results are based on the initial measured test item concentrations.

Results

The test item had a statistically significant inhibitory effect on the growth (growth rate and yield) of Pseudokirchneriella subcapitata after the test period of 72 hours at the initial measured test concentrations of 0.040 mg/L and above. Thus, this test concentration was determined as the 72-hour LOEC. The 72-hour NOEC was determined to be 0.022 mg/L, since up to and including this test concentration the mean growth rate and yield of the algae were not statistically significantly lower than in the control. The EC values as well as the NOEC and LOEC were calculated for both parameters, the growth rate (μ) and yield (y) the after 72 hours test duration on the basis of initial measured test item concentrations.

Description of key information

The test item had a statistically significant inhibitory effect on the growth (growth rate and yield) of Pseudokirchneriella subcapitata after the test period of 72 hours at the initial measured test concentrations of 0.040 mg/L and above. Thus, this test concentration was determined as the 72-hour LOEC. The 72-hour NOEC was determined to be 0.022 mg/L, since up to and including this test concentration the mean growth rate and yield of the algae were not statistically significantly lower than in the control.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.084 mg/L

EC10 or NOEC for freshwater algae:

0.022 mg/L

Additional information

Introduction

The influence of the test item 1H-Imidazole-1-ethanol, 4,5-dihydro, 2-nortall-oil, alky derivatives on the growth of the freshwater green algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) was investigated in a 72-hour static test according to the EU Commission Directive 92/69/EEC, C.3 (1992), and the OECD Guideline 201 (2006).

Methods

Exponentially growing cultures of this unicellular algal species were exposed to various concentrations of the test item under defined conditions. A static, non-renewal exposure system was used. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours and, thus, over several algal generations. The nominal test concentrations were 0.010, 0.022, 0.046, 0.10, 0.22, and 0.46 mg/L in parallel to a control.

At the start of the test, the measured test item concentrations in the analyzed test media ranged from 75 to 100% of the nominal values. At the end of the test, 84 to 98% of the initial measured values were found. Thus, the test item was sufficiently stable during the test period and the reported biological results are based on the initial measured test item concentrations.

Results

The test item had a statistically significant inhibitory effect on the growth (growth rate and yield) of Pseudokirchneriella subcapitata after the test period of 72 hours at the initial measured test concentrations of 0.040 mg/L and above. Thus, this test concentration was determined as the 72-hour LOEC. The 72-hour NOEC was determined to be 0.022 mg/L, since up to and including this test concentration the mean growth rate and yield of the algae were not statistically significantly lower than in the control. The EC values as well as the NOEC and LOEC were calculated for both parameters, the growth rate (μ) and yield (y) the after 72 hours test duration on the basis of initial measured test item concentrations.