Chlorothalonil

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:

3 Aug 1998 to 8 Aug 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Qualifier:

according to guideline

Guideline:

other: Pesticide Assessment Guidelines Subdivision J Hazard Evaluation: Nontarget Plants. EPA 540/09-82-020

Version / remarks:

1982

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

At the start of the test, samples were taken of each test solution, using the excess remaining after filling the test vessels. At the end of the test each blank solution was sampled and analysed in the same manner.

Vehicle:

yes

Remarks:

dimethylformamide

Details on test solutions:

A 560 mg/L nominal concentration primary stock solution of the test substance was prepared by dissolving 0.0056 g in 10 mL of dimethylformamide. A further intermediate stock solution, 70 mg/L, was prepared in dimethylformamide using an aliquot of the primary stock solution. Each test solution was prepared by the addition of an aliquot of the relevant stock solution to sterile culture medium. These introductions were made, post equalising additions of dimethylformamide, where applicable, such that the solvent control and all test concentrations contained 0.10 mL dimethylformamide/L. The control consisted of culture medium only. All additions were made sub-surface, gradually, by microlitre syringe, into solutions vigorously stirred by magnetic follower. After preparation, a visual assessment of the test solutions showed them all to be clear and colourless. Using aseptic techniques, 100 mL volumes of the appropriate test solution were dispensed to each test and blank vessel, with the remainder of the test solutions being used for physical and chemical analyses.

Test organisms (species):

Navicula pelliculosa

Details on test organisms:

TEST ORGANISM
- Common name: Diatom
- Strain: UTEX 667
- Source: Laboratory cultures maintained under axenic conditions
- Age of inoculum: 3 day old culture of the diatom in the exponential growth phase
- Culturing media and conditions: Same as the test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

120 h

Test temperature:

- 24 ± 1 °C (hourly temperature measurement)
- 23.8 - 24.0 °C (daily temperature measurement)

pH:

- Start of the test: 8.6 - 8.7
- End of the test: 7.7 - 7.8
- No increases of pH were observed over the test duration.

Nominal and measured concentrations:

- Nominal concentration: 0 (culture medium control), 0 (solvent control), 0.90, 1.8, 3.5, 7.0, 14, 28 and 56 µg/L
- Measured concentration: < 0.060, < 0.060, 1.1, 2.2, 3.9, 7.5, 14, 31 and 59 µg/L, respectively. See Table 1 in 'Any other information on materials and methods incl. tables'

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL borosilicate glass conical flasks
- Type: Closed with polyurethane foam bungs
- Filled volume: 100 ml of test solution
- Shaking: Approximately 140 rpm
- Mean measured initial cells density: 0.301E+04 cells mL/L
- No. of vessels per concentration: 3
- No. of vessels per control: 3
- No. of vessels per vehicle control: 6
- The positions of the test vessels in the incubator were randomised by rows, and re-randomised daily.
- One blank vessel (without diatom inoculum) for each control and test concentration was incubated concurrently.
- Light source: "Cool-white" illumination
- Light intensity: 4300 lux (50.6 µ einsteins/m2/s)

GROWTH MEDIUM
- Standard medium used: No
- Detailed composition if non-standard medium was used: See Table 2 in "Any other information on materials and methods incl. tables"

TEST MEDIUM / WATER PARAMETERS
- pH: The pH of each test solution was measured at the start of the test, using the excess remaining after filling the test vessels. At the end of the test the pH of two of the replicate test solutions (containing diatoms) from the dilution water control and each test concentration was determined.
- Temperature: The temperature of the incubator was measured daily by a thermometer calibrated to 0.1°C. It was also continuously monitored, with hourly recording of values, using an automatic recording system linked to a thermistor.
- Light: The light intensity was measured once during the study.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The diatom cell densities of the inoculum and test cultures were determined by electronic particle counting, using a coulter counter, counting at a lower threshold equivalent spherical diameter of approximately 2.3 µm.
- After 1, 2, 3, 4 and 5 days (24, 48, 72, 96 and 120 hours), samples were removed from each test and blank vessel. The appropriate blank particle count was subtracted from that of the test culture to obtain the cell density.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

120 h

Dose descriptor:

NOEC

Effect conc.:

3.5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

120 h

Dose descriptor:

EC50

Effect conc.:

27 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

120 h

Dose descriptor:

NOEC

Effect conc.:

3.5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

120 h

Dose descriptor:

EC50

Effect conc.:

8.8 µg/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 3, 4 and 5 in 'Any other information on results incl. tables'

CELL DENSITY
The cell density was determined by coulter counter daily. At the start of the test, the initial cell density was 0.301E+04 cells ml/L. After 3 days (72-hours) of exposure, cell density in the culture medium control averaged approximately 1.48E+05 cells/mL and in the solvent control average approximately 1.75E+05 cells/mL. On day 3 (72-hour), the averaged algal density in the 0.90, 1.8, 3.5, 7.0, 14, 28 and 56 µg/L treatment levels were 1.66, 1.46, 1.25, 0.62, 0.54, 0.0351, 0.0345E+05 cells/mL, respectively. Following 5 days (120-hour) of exposure, cell biomass in the culture medium negative control averaged 1.32E+06 cells/mL and in the solvent control average approximately 1.42E+06 cells/mL. The 0.90, 1.8, 3.5, 7.0, 14, 28 and 56 µ/L treatment levels showed a 1.51, 1.42, 1.40, 0.92, 0.86, 0.0391 and 0.0092E+06 cells/mL algal density, respectively, on day 5 (at 120-hour).

GROWTH RATE
After 72 hours of exposure, the growth rate of algae in culture medium control was 96% of it does in the solvent control. In comparing with the solvent control, the growth rate of diatom in 0.90, 1.8, 3.5, 7.0, 14, 28 and 56 µg/L treatments were 99%, 96%, 92%, 74%, 71%, 4% and 3% respectively. After 120 hours of exposure, the growth rate of algae in culture medium control remained 99% of the solvent control. In comparing with the solvent control, the growth rate of diatom in 0.90, 1.8, 3.5, 7.0, 14, 28 and 56 µg/L treatments were 101%, 100%, 100%, 93%, 92%, 42% and 18% respectively.

NOEC and EC50
Based on the results of biomass, the 72-h NOEbC was 1.8 µg/L, LOEbC was 3.5 µg/L and EbC50 was 5.1 µg/L (95% confidence limits 4.5 – 5.8 µg/L). Based on the results of growth rate, the 72-h NOErC was 3.5 µg/L, LOErC was 7.0 µg/L and ErC50 was 13 µg/L (95% confidence limits 11 – 14 µg/L).

Table 3. Diatom cell density

Nominal concentration (µg/L)	Replicate	Diatom cell density (x 104 cells/mL)
		Day 1	Day 2	Day 3	Day 4	Day 5
Culture medium control	A	0.994	3.17	13.2	48.1	124
	B	0.820	3.37	16.4	54.4	144
	C	0.922	3.18	14.7	42.3	119
	Mean	0.912	3.24	14.8	48.3	132
Solvent control	A	0.783	4.07	16.0	54.9	131
	B	0.889	4.35	18.4	64.2	161
	C	0.797	3.73	14.1	41.7	110
	D	0.732	4.54	20.1	73.4	169
	E	0.793	3.94	17.7	61.0	138
	F	0.764	4.25	18.5	64.1	145
	Mean	0.793	4.15	17.5	59.9	142
0.90	A	0.726	3.73	15.0	51.0	130
	B	0.691	4.10	18.4	62.9	152
	C	0.732	3.63	16.5	65.6	171
	Mean	0.716	3.82	16.6	59.8	151
1.8	A	0.694	4.07	15.6	49.1	136
	B	0.658	3.30	13.8	55.3	171
	C	0.699	4.12	14.3	46.1	120
	Mean	0.684	3.83	14.6	50.2	142
3.5	A	0.538	2.65	11.6	47.3	123
	B	0.494	3.01	15.0	67.2	179
	C	0.582	2.67	11.0	46.4	118
	Mean	0.538	2.78	12.5	53.6	140
7.0	A	0.260	1.67	8.57	39.1	115
	B	0.367	1.27	5.40	25.9	89.1
	C	0.283	1.07	4.62	22.6	72.3
	Mean	0.303	1.34	6.20	29.2	92
14	A	0.274	1.45	6.50	28.7	103
	B	0.233	0.730	4.47	20.5	76.8
	C	0.269	1.10	5.24	23.4	78.9
	Mean	0.259	1.09	5.40	24.2	86.0
28	A	0.044	0.251	0.398	1.11	4.21
	B	0.030	0.246	0.295	0.912	4.41
	C	0.019	0.404	0.359	0.960	3.12
	Mean	1.031	0.300	0.351	0.99	3.91
56	A	0.152	0.305	0.457	0.912	1.19
	B	0.146	0.264	0.307	0.502	0.770
	C	0.195	0.340	0.271	0.618	0.794
	Mean	0.164	0.303	0.345	0.677	0.92

 

 

Table 4. Mean area under the growth curve indicating statistically significant differences, together with percentages of the solvent control

Nominal concentration (µg/L)	0 – 72 hour		0 – 96 hour		0 – 120 hour
	Mean area under growth curve	% of solvent control	Mean area under growth curve	% of solvent control	Mean area under growth curve	% of solvent control
Culture medium control	10.8 *	83	42.0	82	132.0	87
Solvent control	12.9	-	51.3	-	152.1	-
0.90	12.1	94	50.0	98	155.1	102
1.8	11.0	85	43.1	84	139.1	91
3.5	8.8*	68	41.6	81	138.1	91
7.0	4.0*	31	21.4*	42	81.7*	54
14	3.3*	26	17.8*	35	72.7*	48
28	- 0.2*	- 2	0.1*	0	2.3*	1
56	- 0.1*	- 1	0.1*	0	0.6*	0

* Significant difference (P=0.05) from the solvent control

 

Table 5. Mean growth rates of the test diatom during the test period, indicating statistically significant differences together with percentages of the solvent control.

Nominal concentration (µg/L)	0 – 72 hour		0 – 96 hour		0 – 120 hour
	Mean area under growth curve	% of solvent control	Mean area under growth curve	% of solvent control	Mean area under growth curve	% of solvent control
Culture medium control	1.298	96	1.270	96	1.217	99
Solvent control	1.354	_	1.323	-	1.232	-
0.90	1.337	99	1.323	100	1.244	101
1.8	1.293	96	1.279	97	1.232	100
3.5	1.294	92	1.296	98	1.228	100
7.0	1.008*	74	1.144*	86	1.145*	93
14	0.963*	71	1.097*	83	1.132*	92
28	0.051*	4	0.299*	23	0.513*	42
56	0.045*	3	0.203*	15	0.223*	18

* Significant difference (P=0.05) from the solvent control

 

Validity criteria fulfilled:

not specified

Conclusions:

Based on the findings, the 72-h NOErC was determined to be 3.5 µg/L and ErC50 was determined to be 13 µg/L.

Executive summary:

The influence of the test substance (98.1%) on the growth of the freshwater diatom, Navicula pelliculosa, was investigated in a 5-day (120-hour) static test in accordance with EPA Pesticide Assessment Guidelines and in compliance with GLP criteria. The test substance was first dissolved in solvent dimethylformamide before further diluting in the test medium. The diatom were exposed to measured concentrations of < 0.060, < 0.060, 1.1, 2.2, 3.9, 7.5, 14, 31 and 59 µg/L (measured by GC, nominal concentrations were 0.90, 1.8, 3.5, 7.0, 14, 28 and 56 µg/L, respectively). But all the results were reported in nominal concentration. In addition, a culture medium control and a solvent control were included in this study as well. 

All treatments were with 3 replicates, except solvent control (with 6 replicates). The test was carried out in an incubator with a temperature range of 24 ± 1°C. Flasks were manually shaken at a speed of 140 rpm. Illumination of 4300 lux (50.6 µ einsteins/m2/s) was provided. Flasks in the incubator were randomised by rows, and re-randomised daily. The pH of the test started from a range of 8.6 – 8.7 and ended with a range of 7.7 – 7.8. Biomass of the algae was determined by cell counts daily using a coulter counter.

The cell density was determined by coulter counter daily. At the start of the test, the initial cell density was 0.301E+04 cells ml/L. After 3 days (72-hours) of exposure, cell density in the culture medium control averaged approximately 1.48E+05 cells/mL and in the solvent control average approximately 1.75E+05 cells/mL. On day 3 (72-hour), the averaged algal density in the 0.90, 1.8, 3.5, 7.0, 14, 28 and 56 µg/L treatment levels were 1.66E+05, 1.46E+05, 1.25E+05, 0.62E+05, 0.54E+05, 0.351E+04, 0.345E+04 cells/mL, respectively. Following 5 days (120-hour) of exposure, cell biomass in the culture medium negative control averaged 1.32E+06 cells/mL and in the solvent control average approximately 1.42E+06 cells/mL. The 0.90, 1.8, 3.5, 7.0, 14, 28 and 56 µ/L treatment levels showed a 1.51E+06, 1.42E+06, 1.40E+06, 0.92E+06, 0.86E+06, 0.391E+05 and 0.92E+04 cells/mL algal density, respectively, on day 5 (at 120-hour).

After 72 hours of exposure, the growth rate of algae in culture medium control was 96% of it does in the solvent control. In comparing with the solvent control, the growth rate of diatom in 0.90, 1.8, 3.5, 7.0, 14, 28 and 56 µg/L treatments were 99%, 96%, 92%, 74%, 71%, 4% and 3% respectively. After 120 hours of exposure, the growth rate of algae in culture medium control remained 99% of the solvent control. In comparing with the solvent control, the growth rate of diatom in 0.90, 1.8, 3.5, 7.0, 14, 28 and 56 µg/L treatments were 101%, 100%, 100%, 93%, 92%, 42% and 18% respectively.

Based on the findings, the 72-h NOErC was determined to be 3.5 µg/L and ErC50 was determined to be 13 µg/L.

Description of key information

Freshwater 72-h NOErC = 3.5 µg/L, Navicula pelliculosa, growth rate, OECD TG 201, Smyth 1998

Freshwater 72-h ErC50 = 13 µg/L, Navicula pelliculosa, growth rate, OECD TG 201, Smyth 1998

Key value for chemical safety assessment

EC50 for freshwater algae:

13 µg/L

EC10 or NOEC for freshwater algae:

3.5 µg/L

Additional information

The influence of the test substance (98.1%) on the growth of the freshwater diatom, Navicula pelliculosa, was investigated in a 5-day (120-hour) static test in accordance with EPA Pesticide Assessment Guidelines and in compliance with GLP criteria. The test substance was first dissolved in solvent dimethylformamide before further diluting in the test medium. The diatom were exposed to measured concentrations of < 0.060, < 0.060, 1.1, 2.2, 3.9, 7.5, 14, 31 and 59 µg/L (measured by GC, nominal concentrations were 0.90, 1.8, 3.5, 7.0, 14, 28 and 56 µg/L, respectively). But all the results were reported in nominal concentration. In addition, a culture medium control and a solvent control were included in this study as well. 

All treatments were with 3 replicates, except solvent control (with 6 replicates). The test was carried out in an incubator with a temperature range of 24 ± 1°C. Flasks were manually shaken at a speed of 140 rpm. Illumination of 4300 lux (50.6 µ einsteins/m2/s) was provided. Flasks in the incubator were randomised by rows, and re-randomised daily. The pH of the test started from a range of 8.6 – 8.7 and ended with a range of 7.7 – 7.8. Biomass of the algae was determined by cell counts daily using a coulter counter.

The cell density was determined by coulter counter daily. At the start of the test, the initial cell density was 0.301E+04 cells ml/L. After 3 days (72-hours) of exposure, cell density in the culture medium control averaged approximately 1.48E+05 cells/mL and in the solvent control average approximately 1.75E+05 cells/mL. On day 3 (72-hour), the averaged algal density in the 0.90, 1.8, 3.5, 7.0, 14, 28 and 56 µg/L treatment levels were 1.66E+05, 1.46E+05, 1.25E+05, 0.62E+05, 0.54E+05, 0.351E+04, 0.345E+04 cells/mL, respectively. Following 5 days (120-hour) of exposure, cell biomass in the culture medium negative control averaged 1.32E+06 cells/mL and in the solvent control average approximately 1.42E+06 cells/mL. The 0.90, 1.8, 3.5, 7.0, 14, 28 and 56 µ/L treatment levels showed a 1.51E+06, 1.42E+06, 1.40E+06, 0.92E+06, 0.86E+06, 0.391E+05 and 0.92E+04 cells/mL algal density, respectively, on day 5 (at 120-hour).

After 72 hours of exposure, the growth rate of algae in culture medium control was 96% of it does in the solvent control. In comparing with the solvent control, the growth rate of diatom in 0.90, 1.8, 3.5, 7.0, 14, 28 and 56 µg/L treatments were 99%, 96%, 92%, 74%, 71%, 4% and 3% respectively. After 120 hours of exposure, the growth rate of algae in culture medium control remained 99% of the solvent control. In comparing with the solvent control, the growth rate of diatom in 0.90, 1.8, 3.5, 7.0, 14, 28 and 56 µg/L treatments were 101%, 100%, 100%, 93%, 92%, 42% and 18% respectively.

Based on the findings, the 72-h NOErC was determined to be 3.5 µg/L and ErC50 was determined to be 13 µg/L.