cyproconazole (ISO); (2RS,3RS;2RS,3SR)-2-(4-chlorophenyl)-3-cyclopropyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Sep 1986 to 7 November 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

June 7, 1984

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples of 5.0 mL each were taken 0 and 96 hours after exposure from untreated, 0.125, 0.5 and 1.0 mg/L test substance treated algae suspensions. Until analysis, the samples were stored at - 20 °C.

Vehicle:

no

Details on test solutions:

Not reported

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The algae were grown in the test facility under standardised conditions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

20 ± 2 ˚C

pH:

7.2 - 8.2

Nominal and measured concentrations:

- Nominal concentrations: 0 (negative control), 0.032, 0.063, 0.125, 0.250, 0.5 and 1.0 mg/mL
- Measured concentrations: Mean measured concentrations were determined for 0.125, 0.50 and 1.0 mg/L treatments resulting in 0.094, 0.451 and 0.939 mg/mL, respectively

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks
- Type: Stoppered with cotton wool plugs
- Shaking during incubation: 120 strokes/min
- Initial cells density: 10,000 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 3

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity: 8000 Lux
- Light source: Eight fluorescent tubes, installed above the shaking water bath in a distance from the algae flasks of about 20 cm.

EFFECT PARAMETERS MEASURED
- Sampling intervals: Samples of algae were taken after 24, 48, 72 and -96 hours of incubation.
- Determination of cell concentrations: The number of algae after 24, 48, 72 and 96 hours was determined spectrophotometrically
on a spectrophotometer by reading the absorption at 700 nm and using a standard curve established with known (counted) cell density.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.077 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

96 h

Dose descriptor:

EC0

Effect conc.:

0.021 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

An overview of the results is provided in Table 2 in ‘Any other information on results incl. tables’.

DENSITY
At the start of the test, the initial algal density was 1.0E+4 cells/mL Following 72-hours of exposure, the cell density in the control averaged as 1.3E+06. The 72-hour cell density in the 0.032, 0.063, 0.125, 0.250, 0.50 and 1.0 mg/L treatment levels were 9.84E+05, 4.88E+05, 2.65E+05, 2.07E+05, 1.44E+05 and 1.27E+05 cells/mL, respectively. After 96 hours treatment, the cell density in the control averaged as 2.45E+06. The average cell density in the 0.032, 0.063, 0.125, 0.250, 0.50 and 1.0 mg/L treatment levels were 2.33E+06, 1.44E+06, 4.69E+05, 2.92E+05, 2.10E+05 and 1.93E+05 cells/mL, respectively.

AUC
The biomass integrals were determined by the areas under the growth curves of algal cultures with different concentrations of the test item. Following 96-hours of exposure, mean area under the growth curve in the control was 7.12E+07. The 96-hour mean area under the growth curve in the 0.032, 0.063, 0.125, 0.250, 0.50 and 1.0 mg/L treatment levels were 6.037E+07, 3.617E+07, 1.864E+07, 1.49E+07, 1.061E+07, and 1.007E+07, respectively.

Results with reference substance (positive control):

An overview of the results is provided in Table 3 in ‘Any other information on results incl. tables’.
DENSITY
At the start of the test, the initial algal density was 1.0E+4 cells/mL Following 72-hours of exposure, the cell density in the control averaged as 7.3E+05. The 72-hour cell density in the 0.6, 1.0, 1.4, 1.8 and 2.2 mg/L treatment levels were 5.47E+05, 3.26E+05, 1.86E+05, 1.15E+05 and 7.06E+04 cells/mL, respectively. After 96 hours treatment, the cell density in the control averaged as 2.13E+07. The average cell density in the 0.6, 1.0, 1.4, 1.8 and 2.2 mg/L treatment levels were 1.776E+06, 1.08E+06, 5.783E+05, 3.064E+05 and 1.195E+05 cells/mL, respectively.

AUC
The biomass integrals were determined by the areas under the growth curves of algal cultures with different concentrations of the test item. Following 96-hours of exposure, mean area under the growth curve in the control was 4.6E+07. The 96-hour mean area under the growth curve in the the 0.6, 1.0, 1.4, 1.8 and 2.2 mg/L treatment levels were 3.7E+07, 2.3E+07, 1.3E+07, 7.0E+06 and 5.0E+06, respectively. The 96h EbC50 of potassium dichromate was 0.998 mg/L.

Table 2. Influence of the test substance on algae growth. (Values given represent mean of triplicate

samples).

Concentration (mg/L)	Number of cell a				AUC b	Inhibition %
	24 h	48 h	72 h	96 h
0 (control)	10.0	30.6	133.5	245.1	7120	-
0.032	10.8	25.9	98.4	232.9	6037	15.2
0.063	9.9	19.9	48.8	144.2	3617	49.2
0.125	11.1	16.6	26.5	46.9	1864	73.8
0.250	11.9	14.9	20.7	29.2	1490	79.1
0.50	8.8	10.5	14.4	21.0	1061	85.1
1.0	9.1	10.5	12.7	19.3	1007	85.9

a. Number of cells per ml divided by 10000 which were determined after 24, 48, 72 and 96 hours, respectively.

b. Represents area under the curve divided by 10000 and corrected for initial cell density = 10000 per mL.

Table 3. Influence of potassium dichromate on algal growth. (Values given represent mean of triplicate samples).

Concentration (mg/L)	Number of cell a				AUC b	Inhibition %
	24 h	48 h	72 h	96 h
0 (control)	1.15	12.53	73.00	213.20	4600	-
0.6	1.19	10.36	54.73	117.60	3700	19.6
1.0	0.91	7.61	32.63	108.00	2300	50.0
1.4	0.83	5.22	18.63	57.83	1300	71.7
1.8	0.67	2.90	11.50	30.64	700	84.8
2.2	0.71	2.47	7.06	19.15	500	89.1

a. Number of cells per ml divided by 10000 which were determined after 24, 48, 72 and 96 hours, respectively.

b. Represents area under the curve divided by 10000 and corrected for initial cell density = 10000 per mL.

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the 96-h EbC0 was calculated to be 0.021 mg/L and the 96-h EbC50 was calculated to be 0.077 mg/L.

Executive summary:

The influence of the test substance on the growth of the freshwater algae, Desmodesmus subspicatus, was investigated in a 96-hour static test following OECD TG 201. The study was conducted in compliance with GLP criteria. The algae were exposed to nominal concentrations of 0.032, 0.063, 0.125, 0.250, 0.5 and 1.0 mg/mL (mean measured concentrations were determined for 0.125, 0.50 and 1.0 mg/L treatments, resulting in 0.094, 0.451 and 0.939 mg/mL, respectively, corresponding to 75.2%, 90.2% respectively 93.9% of nominal; measured by GLC). In addition, a negative control and a positive control (potassium dichromate, five concentrations) were included in this study as well. Three replicates per test concentration and control each containing an initial algal density was 1.0E+04 cells/mL were used. The test was carried out under the following conditions: 20 °C, 8000 lux light intensity at the level of the test solutions, pH 7.2 – 8.2. Small volumes (2-5 mL) of all test concentrations and controls were taken from all test flasks after 24, 48, 72 and 96 hours of exposure. The algal cell densities in these samples were determined by a spectrophotometer (absorption at 700 nm).

In the results, a clear dose-responsive toxicity profile is observed. After 72 hours, the cell density in the control averaged as 1.3E+06. The 72-hour cell density in the 0.032, 0.063, 0.125, 0.250, 0.50 and 1.0 mg/L treatment levels were 9.84E+05, 4.88E+05, 2.65E+05, 2.07E+05, 1.44E+05 and 1.27E+05 cells/mL, respectively. After 96-hours of exposure, mean area under the growth curve in the control was 7.12E+07. The 96-hour mean area under the growth curve in the 0.032, 0.063, 0.125, 0.250, 0.50 and 1.0 mg/L treatment levels were 6.037E+07, 3.617E+07, 1.864E+07, 1.49E+07, 1.061E+07, and 1.007E+07, respectively. Based on the findings, the 96-h EbC0 was graphically determined to be 0.021 mg/L and the 96-h EbC50 was calculated to be 0.077 mg/L.

Description of key information

All available data was assessed and the studies representing the worst-case effects are included here as key. The results can be considered worst-case and are selected for the CSA. Other studies are included as supporting information.

Freshwater, 96-h EbC0 = 0.021 mg/L, static, Desmodesmus subspicatus, OECD TG 201, Ellgehausen 1986

Freshwater, 96-h EbC50 = 0.077 mg/L, static, Desmodesmus subspicatus, OECD TG 201, Ellgehausen 1986

Key value for chemical safety assessment

EC50 for freshwater algae:

0.077 mg/L

EC10 or NOEC for freshwater algae:

0.021 mg/L

Additional information

There are three standard guideline followed and GLP compliant studies available for this endpoint. The 96-h study on freshwater green algae (Desmodesmus subspicatus) is selected to be the key study, because it represents the worst-case effects (i.e. showed the lowest effect value) for freshwater conditions. The algae were exposed to nominal concentrations of 0.032, 0.063, 0.125, 0.250, 0.5 and 1.0 mg/mL (mean measured concentrations were determined for 0.125, 0.50 and 1.0 mg/L treatments, resulting in 0.094, 0.451 and 0.939 mg/mL, respectively; measured by GLC). In addition, a negative control  was  included in this study as well. Three replicates per test concentration and control each containing an initial algal density  of 1.0E+04 cells/mL  was used. Small volumes (2 - 5 mL) of all test concentrations and controls were taken from  all test flasks after 24, 48, 72 and 96 hours of exposure. The algal cell densities in these samples were determined by a spectrophotometer (absorption at 700 nm).

After 96-hours of exposure, mean area under the growth curve in the control was 7.12E+07. The 96-hour mean area under the growth curve in the 0.032, 0.063, 0.125, 0.250, 0.50 and 1.0 mg/L treatment levels were 6.037E+07, 3.617E+07, 1.864E+07, 1.49E+07, 1.061E+07, and 1.007E+07, respectively. Based on  nominal concentrations, the 96-h EbC0 was graphically determined to be 0.021 mg/L and the 96-h EbC50 was calculated to be 0.077 mg/L (Ellgehausen 1986, OECD TG 201, Reliability 2).

 

The other two studies are supporting studies. The first supporting study was performed with the freshwater green algae Raphidocelis subcapitata. The algae were exposed to nominal concentrations of 0.010, 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L (mean measured concentrations were 0.00894, 0.0300, 0.0972, 0.304, 0.972, 3.02 and 10.0 mg/L, respectively). Based on nominal concentrations, the 72-h NOErC was derived to be 0.032 mg/L. The effects of the test substance on growth rate inhibition were not significant enough to determine a reliable ErC50, which was therefore derived to be >10 mg/L (Weber 2010, OECD TG 201, Reliability 1). The second supporting study was performed with the freshwater green algae Chlorella vulgaris. The algae were exposed to nominal test substance concentrations of 0 (control), 50, 100, 200, 400, 800 and 1600 µg/L (mean measured: ND, 45.9, 97.5, 196, 392, 820 and 1530 µg/L, respectively). Based on mean measured concentrations, the 72-hour NOErC was determined to be 392 µg/L and the 72-hour ErC50 was determined to be 1176 µg/L (Jenkins 1993, OECD TG 201, Reliability 1).