pymetrozine (ISO); (E)-4,5-dihydro-6-methyl-4-(3-pyridylmethyleneamino)-1,2,4-triazin-3(2H)-one

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:

25 May 1995 to 08 Jun 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: US EPA FIFRA Guideline 123-2

Version / remarks:

1989

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Analytical determination of the test substance concentration (active ingredient) was performed with 40 mL samples collected from each test concentration prior to its addition into the triplicate test vessels at the beginning of the test. Approximately 13 mL from each replicate test vessel was sampled at the end of the test and replicate samples were pooled prior to analysis. samples were stored in 40 mL amber glass bottles. samples or extracts were refrigerated if not analysed on the day of collection. Analysis of the test substance at 0 and at 120 hour.

Vehicle:

no

Remarks:

sterile synthetic media

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A 100 mg/L stock solution was formulated by adding 0.0509 g test substance to sterile synthetic media and adjusting the final volume to 500 mL. The stock solution was sonicated to aid in the dissolution of the test substance. Appropriate amounts of this stock solution were added directly to sterile media to formulate test concentrations.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Age of inoculum (at test initiation): 8 days

ACCLIMATION
- Acclimation period: 14 day
- Culturing media and conditions: same as the test
- Any deformed or abnormal cells observed: no abnormal cells observed. Culture was actively growing at the start of the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

120 h

Test temperature:

23.4 – 23.8 °C

pH:

- pH at study initiation: 7.5
- pH at study termination: 7.9 - 8.9

Nominal and measured concentrations:

Nominal concentrations: 0 mg/L (control), 6.3, 13, 25, 50, and 100 mg/L
Measured concentrations: <2.53 mg/L (control), 6.28, 12.8, 23.2, 47.1, and 96.7 mg/L (see 'Any other information on materials and methods incl. tables').

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL erlenmeyer flasks
- Material, size, headspace, fill volume: 50 mL
- Control end cells density: 1,986E+06 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 3

TEST MEDIUM / WATER PARAMETERS
- Total organic carbon: not detected

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: water used for acclimation of test organisms and for all toxicity testing was sterile enriched media with a target pH of 7.5 ± 0.1.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: A 24 hour light and 0 hour dark photoperiod was automatically maintained
- Light intensity and quality: Cool-white fluorescent lights that provided a light intensity of 3600 lux.

WATER QUALITY PARAMETERS
Temperature of the incubator was measured and recorded daily and pH (Beckman model pHI 12 meter) was determined in each test vessel at the beginning and end of the test. The temperature of a representative flask of water incubated among the test vessels was recorded continuously.

EFFECT PARAMETERS MEASURED: cell density
The number of algal cells/ml in each test vessel was determined microscopically using a hemacytometer after 24, 48, 72, 96, and 120 hours of exposure.

RANGE-FINDING STUDY
A screening test was conducted from April 20 to 25, 1995. The test was performed with a control and 5 concentrations of the test substance (0.010, 0.10, 1.0, 10, and 100 mg/L). After 120 hours there was <1% of control growth at 100 mg/L, and at least 88% of the control growth at 0,010, 0.10, 1.0, and 10 mg/L.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.28 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

23.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence intervals: 15.0 to 37.8 mg/L

Duration:

72 h

Dose descriptor:

other: EC25

Effect conc.:

6.87 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence intervals: 3.23 to 14.6 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

6.28 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

16.9 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence intervals: 9.89 to 28.7 mg/L

Duration:

96 h

Dose descriptor:

other: EC25

Effect conc.:

5.86 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence intervals: 2.58 to 13.3 mg/L

Duration:

120 h

Dose descriptor:

NOEC

Effect conc.:

6.28 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

120 h

Dose descriptor:

EC50

Effect conc.:

21.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence intervals: 11.1 to 42.1 mg/L

Duration:

120 h

Dose descriptor:

other: C25

Effect conc.:

6.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence intervals: 2.22 to 19.1 mg/L

Details on results:

The results are tabulated in 'Any other information on results incl. tables'.
- Test item solubility: Insoluble material was not observed in any test vessel during the test.
- Exponential growth in the control (for algal test): yes, the algal population in the control vessels grew well, resulting in an average of 1,986,000 cells/mL after 120 hours. The slope of the 120 hour dose response curve is 1.6.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Because measured concentrations at 120 hours were all less than 70% of nominal concentrations, initial measured concentrations were used for all calculations

Reported statistics and error estimates:

All statistical analyses were performed using the initial measured concentrations of the test substance. The weighted least squares non-linear regression method was used to calculate EC25 and EC50 values and 95% confidence intervals (Bruce and Versteeg, 1992). The no observed effect concentration (NOEC) for algal growth was statistically calculated (TOXSTAT; Gulley, et. al., 1990). A Shapiro Wilks test was used to determine that data were normally distributed and Bartlett's test was used to determine that variances were homogeneous. Because data were normally distributed and variances were homogeneous, a parametric one-way analysis of variance (ANOVA) and Dunnett's t-test were used to compare treatment and control data. The slope of the 120 dose response curve was calculated using the probit method (Stephan, 1983).

cell growth data from the toxicity test with the test substance and the freshwater alga, Pseudokirchneriella subcapitata

Initial Measured Concentration	Number of Cells/mL (x10E+03)
	Replicate	0h	24h	48h	72h	96h	120h
<2.53 Control	1	3	6	24	166	618	1700
	2	3	10	18	206	842	2162
	3	3	8	28	202	760	2096
	Mean	3	8	23	191	740	1986
	%control
6.28	1	3	8	20	168	782	1980
	2	3	8	24	172	650	2206
	3	3	8	18	182	746	2118
	Mean	3	8	2	174	726	2101
	%control	100	100	91	91	98	106
12.8	1	3	6	118	118	304	1108
	2	3	6	122	122	380	1012
	3	3	10	102	102	322	758
	Mean	3	7	114	114	335	959
	%control	100	88	60	60	45	48
23.2	1	3	8	92	92	278	1010
	2	3	6	104	104	306	950
	3	3	8	74	74	290	942
	Mean	3	788	90	90	291	967
	%control	100	6	47	47	39	49
47.1	1	3	6	72	72	212	620
	2	3	6	50	50	182	518
	3	3	8	82	82	232	618
	Mean	3	7	68	68	209	585
	%control	100	88	36	36	28	29
96.7	1	3	<3	48	48	93	564
	2	3	<3	62	62	158	528
	3	3	<3	38	38	106	526
	Mean	3	<3	49	49	119	539
	%control	100	<38	26	26	16	27

Validity criteria fulfilled:

not specified

Conclusions:

The 72-h ErC50 and NOErC were determined to be 23.8 mg/L (15.0 to 37.8 mg/L) and 6.28 mg/L, respectively.

- RAR interpretation statement:
In a toxicity test in freshwater algae, the NOEC of the test subatstance amounts to 6.28 mg/L for all endpoints and assessment intervals ≥ 72 hours. Risk of the test substance to aquatic organisms is clearly not determined by its toxicity to algae.

Executive summary:

The toxicity of the test substance to the freshwater alga, Pseudokirchneriella subcapitata, was investigated, in accordance with US EPA FIFRA Guideline 123-2 and in compliance with GLP-criteria. The test, which was designed to establish the 72, 96, and 120 hour EC25s and EC50s, and no observed effect concentrations (NOECs), was performed under static conditions with five concentrations of test substance and a dilution water control at a temperature of 24 ± 2°C. The dilution water was sterile enriched media with a pH of 7.5. cell counts were determined microscopically using a hemacytometer after 24, 48, 72, 96, and 120 hours of exposure. Nominal concentrations of the active ingredient of the test substance were: 0 mg/L (control), 6.3, 13, 25, 50, and 100 mg/L. Measured concentrations of the test substance ranged from 93 to 100% of nominal values at the start of the test. Initial measured concentrations were <2.53 mg/L (LOD; control), 6.28, 12.8, 23.2, 47.1, and 96.7 mg/L. Because measured concentrations at 120 hours were all less than 70% of nominal concentrations, initial measured concentrations were used for all calculations. The exposure of Pseudokirchneriella subcapitata to the test substance for 120 hours resulted in a median effective concentration (EC50) of 21.6 mg/L, with a 95% confidence interval of 11.1 to 42.1 mg/L. The 120 hour no observed effect concentration (NOEC) was 6 .28 mg/L. Further. the exposure of Pseudokirchneriella subcapitata to the test substance for 72 hours resulted in a median effective concentration (EC50) of 23.8 mg/L, with a 95% confidence interval of 15.0 to 37.8 mg/L. The 72 hour no observed effect concentration (NOEC) was 6.28 mg/L.

Description of key information

All available data was assessed and the studies representing the worst-case effects were included as weight-of-evidence studies. Other studies are included as supporting information. The weight-of-evidence studies are considered to be worst-case and were selected for the CSA.

- 72-h ErC50 = 23.8 mg/L and NOErC = 6.28 mg/L in aquatic algae (Pseudokirchneriella subcapitata), US EPA FIFRA Guideline 123-2, Boeri 1995.

Key value for chemical safety assessment

EC50 for freshwater algae:

23.8 mg/L

EC10 or NOEC for freshwater algae:

6.28 mg/L

Additional information

Table: Overview of aquatic algae toxicity data available for the substance:

Species	Guideline / GLP	Endpoint	Effect value	Comment	Reference
Pseudokirchneriella subcapitata	US EPA FIFRA Guideline 123-2 / GLP	72-h ErC50 72-h NOErC	23.8 mg/L 6.28 mg/L	Static regime. Based on nominal concentrations and growth rates.	Boeri, 1995