4-(4-chlorophenyl)-2-phenyl-2-[(1H-1,2,4-triazol-1-yl)methyl]butanenitrile

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-09-26 to 2008-10-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.4400 (Aquatic Plant Toxicity Test using Lemna spp. Tiers I & II))

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The concentration of fenbuconazole in test solutions was measured in samples collected from fresh solutions on days 0, 3, and 5, and from spent solutions on days 3, 5, and 7 of the definitive test. Samples fortified (i.e., QC samples) with fenbuconazole were also prepared for analysis at each sample period.

Vehicle:

yes

Remarks:

Dimethyl formamide

Details on test solutions:

The test medium was 20X-AAP medium. The medium, 20X freshwater algal nutrient medium (20X-AAP),
was prepared by the addition of appropriate reagent grade salts to autoclaved reagent water. Reagent water is produced by passing reverse-osmosis water through a series of deionization tanks, a laboratory water purification system consisting of carbon, de-mineralization, and organic adsorption cartridges, and then through a 0.2-µm filter. After preparation, the medium was adjusted to pH 7.5 ± 0.1 with 0.1N HCl and filtered through 0.45-µm filters.

At test initiation, an 80 mg/mL primary standard was prepared by diluting 0.8122 g (0.8000 g adjusted for purity) of fenbuconazole to a final volume of 10 mL with dimethyl formamide (DMF). Aliquots of this primary standard were used to prepare working standard solutions at concentrations of 1.3, 2.6, 5.0, 10, 20, and 40 mg/L in DMF, each at a volume of 10 mL. Test treatment solutions were prepared individually using 0.050-mL volumes of the working standard solutions and 1-L volumes of 20X-freshwater algal nutrient medium.

The working standards were diluted in the same manner on days 3 and 5 to prepare fresh solutions for the renewals. The final vehicle concentration in each of the exposure concentrations was 50 µL DMF/L. The vehicle control was prepared by adding 0.050 mL of DMF to 1.0 L of the dilution medium resulting in a vehicle concentration of 50 µL DMF/L. The control was comprised of 20X-freshwater algal nutrient medium without test substance.

Test organisms (species):

Lemna gibba

Details on test organisms:

The source of the test species was USDA/ARS Beltsville Agricultural Research Center, Beltsville, Maryland, USA.

Test type:

semi-static

Water media type:

freshwater

Remarks:

20X freshwater algal nutrient medium

Total exposure duration:

7 d

Test temperature:

Temperature of the new and old test solutions ranged from 23.9 to 26.4ºC.
The temperature of the incubator remained within the 25 ± 2ºC range.

pH:

pH of fresh test solutions: 7.5-7.6.

pH of spent test solutions: 8.6-9.1.

Details on test conditions:

A range-finding test was initiated on June 20, 2008, at test concentrations of 0 (control), 0.010, 0.10, 1.0, 10, and 100 mg/L. Each treatment was tested in duplicate and all test and control solutions were renewed on days 3 and 5 of the test. After seven days, the percent inhibition in total number of normal fronds, as compared to the control, was 11, 13, 55, 77, and 77% at nominal concentrations of 0.010, 0.10, 1.0, 10, and 100 mg/L, respectively. Based on the range-finding test results, a nominal concentration range of 0 (control), 0 (vehicle control), 0.13, 0.25, 0.50, 1.0, 2.0, and 4.0 mg/L was selected for the definitive test.

The test chambers used for the test were 500-mL Erlenmeyer flasks with foam stoppers. The control and each test substance treatment were replicated three times and each replicate contained 200 mL of the appropriate test solution. The flasks were randomly positioned on each renewal day and incubated at 25 ± 2ºC for seven days in a temperature-controlled environmental chamber under continuous warm-white fluorescent lighting. The temperature and light intensity within the environmental chamber were measured on days 0 (test initiation), 3, 5, and 7 (test termination). The light intensity was measured with a LI-COR Model LI-189 light meter equipped with a photometric sensor and ranged from 6,140 to 6,393 lux. The temperature of the environmental chamber was monitored continuously during the definitive test using an electronic data-logger.

At test initiation, each test flask received three plants, for a total of 12 fronds. Aseptic addition
of Lemna gibba was initiated and completed within one hour of test solution preparation.
Frond observations and counts were performed on days 3, 5, and 7 for all replicates of the controls and each test substance treatment.

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.237 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

growth rate

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.495 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

growth rate

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

0.835 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.237 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.495 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

3.53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.237 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

dry weight

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.495 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

dry weight

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

1.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

dry weight

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.237 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

growth rate

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.495 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

growth rate

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 4.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

growth rate

Details on results:

The doubling time of total frond number in the control was 1.5 days (corresponding to approximately a 22-fold increase in seven days). The control average specific growth rate for total number of fronds on day 0 to 7 was 0.44/day. The coefficient of variation for the control average specific growth rate from day 0 to day 7, calculated as the standard deviation divided by the mean times 100, was 2%. The coefficient of variation for the control number of total fronds at day 7, calculated as the standard deviation divided by the mean times 100, was 5%. The frond growth in the control over the 7-day test period and the low variability in average specific growth rate between control replicates demonstrated the acceptability of the test.

The percent inhibition of number of total fronds as compared to the pooled control was -5, 8, 13, 34, 62, 74, and 79% for the 0.0645, 0.121, 0.237, 0.495, 1.02, 2.08, and 4.45 mg /L test substance treatments, respectively.

The percent inhibition of total frond average specific growth rate as compared to the pooled control was -1, 3, 5, 14, 32, 44, and 52% for the 0.0645, 0.121, 0.237, 0.495, 1.02, 2.08, and 4.45 mg/L test substance treatments, respectively.

The percent inhibition of total biomass as dry weight as compared to the pooled control was -7, -6, 10, 32, 52, 58, and 61% for the 0.0645, 0.121, 0.237, 0.495, 1.02, 2.08, and 4.45 mg/L test substance treatments, respectively.

The percent inhibition of total biomass average specific growth rate for the test substance treatments, as compared to the pooled control, was -2, -2, 4, 15, 28, 33, and 36% in the 0.0645, 0.121, 0.237, 0.495, 1.02, 2.08, and 4.45 mg/L treatments, respectively.

Table 1

Mean Number of Total Fronds During a 7-Day Exposure of the Duckweed, Lemna gibba, to Fenbuconazole.

Geometric mean measure concentration (mg/L)	Mean number of Total Fronds^a			%Inhibition^b
	Day 3	Day 5	Day 7	Day 7
Control	44	105	258 (CV:5%)	----
Vehicle control	43	105	250 (CV:13%)	----
0.0645	47	105	267 (CV:12%)	-5
0.121	46	108	235 (CV:6%)	8
0.237	44	103	220 (CV: 8%)	13
0.495	45	93	167* (CV: 11%)	34
1.02	37	60*	96* (CV: 13%)	62
2.08	34	53*	66* (CV: 12%)	74
4.45	32*	46*	53* (CV: 15%)	79

^a Values are means of three replicate test chambers.
^b Percent inhibition as compared to the pooled control was determined on day 7 using the following equation:

% inhibition = (((mean number of total fronds in pooled control) - (mean number of total fronds in treatment))/
mean number of total fronds in pooled control) × 100

Key to Abbreviation: CV = coefficient of variance.

Note: On day 0, three plants were placed in each replicate (n = 3) resulting in a total of 12 fronds per replicate.

* Significant growth reduction as compared to the pooled control
(Dunnett’s test, p < 0.05).

 

Table 2

Mean Average Specific Growth Rate of Fronds During a 7-Day Exposure of the Duckweed, Lemna gibba, to Fenbuconazole.

Geometric mean measure concentration (mg/L)	Mean Average Specific Growth Rate (day ^-1) ^a			%Inhibition^b
	Day 0-3	Day 0-5	Day 0-7	Day 0-7
Control	0.434 (CV:10%)	0.433 (CV: 4%)	0.438 (CV:2%)	----
Vehicle control	0.419 (CV: 14%)	0.431 (CV: 8%)	0.433 (CV:4%)	-----
0.0645	0.452 (CV: 3%)	0.434 (CV: 4%)	0.442 (CV:4%)	-1
0.121	0.450 (CV: 4%)	0.439 (CV: 1%)	0.425 (CV:2%)	3
0.237	0.434 (CV: 9%)	0.429 (CV: 4%)	0.415 (CV: 3%)	5
0.495	0.440 (CV: 4%)	0.409 (CV: 2%)	0.376* (CV: 4%)	14
1.02	0.375 (CV: 6%)	0.321* (CV: 7%)	0.296* (CV: 6%)	32
2.08	0.342 (CV: 12%)	0.295* (CV: 7%)	0.243* (CV: 7%)	44
4.45	0.319* (CV:  20%)	0.268* (CV: 12%)	0.211* (CV: 11%)	52

^a Values are means of three replicate test chambers. Values are rounded to three significant figures.

^b Percent inhibition as compared to the pooled control was determined on day 7 using the following equation:

% inhibition = (((mean average specific growth rate of pooled control) - (mean average specific growth
rate of treatment))/ mean average specific growth rate of pooled control) × 100

Key to Abbreviation: CV = coefficient of variance.
* Significant growth reduction as compared to the pooled control (Dunnett’s test, p < 0.05).

 

Table 3

Biomass (Dry Weight) Data for Lemna gibba Exposed to Fenbuconazole.

Geometric mean measure concentration (mg/L)	REP	Day 0 (g)	Day 7 (g)	Treatment mean	%Inhibition^b
Representative sample	A	0.0025	--	0.0022	N/A
	B	0.0023	--
	C	0.0018	--
Control	A	--	0.0307	0.0322	--
	B	--	0.0319
	C	--	0.0341
Vehicle Control	A	--	0.0317	0.0305	--
	B	--	0.0260
	C	--	0.0339
0.0645	A	-	0.0334	0.0335	-7
	B	-	0.0291
	C	-	0.0381
0.121	A	-	0.0341	0.0333	-6
	B	-	0.0305
	C	-	0.0352
0.237	A	-	0.0268	0.0283	10
	B	-	0.0292
	C	-	0.0289
0.495	A	-	0.0207	0.0213*	32
	B	-	0.0206
	C	-	0.0225
1.02	A	-	0.0150	0.0149*	52
	B	-	0.0147
	C	-	0.0151
2.08	A	-	0.0143	0.0133*	58
	B	-	0.0147
	C	-	0.0108
4.45	A	-	0.0086	0.0122*	61
	B	-	0.0132
	C	-	0.0149

^a Yield = day 7 biomass – day 0 mean biomass.
^b Percent inhibition as compared to the pooled control was determined using the following
equation:

% inhibition = (((pooled control mean biomass) - (treatment mean biomass))/ pooled control
mean biomass) × 100

* Significant growth reduction as compared to the pooled control (Dunnett’s test, p < 0.05)

 

Table 4

Biomass (Dry Weight) Average Specific Growth Rate Data for Lemna gibba
Exposed to Fenbuconazole.

Geometric mean measure concentration (mg/L)	Biomass (Dry Weight) Average Specific Growth (Rate per Day^-1)			%Inhibition^a
	Sample (A,B,C)	Days 0-7	Treatment mean
Control	A	0.377	0.384 (CV:2%)	--
	B	0.382
	C	0.392
Vehicle control	A	0.381	0.375 (CV: 5%)	--
	B	0.353
	C	0.391
0.0645	A	0.389	0.388 (CV: 5%)	-2
	B	0.369
	C	0.407
0.121	A	0.392	0.388 (CV: 3%)	-2
	B	0.376
	C	0.396
0.237	A	0.357	0.365 (CV: 2%)	4
	B	0.369
	C	0.368
0.495	A	0.320	0.324* (CV: 2%)	15
	B	0.320
	C	0.332
1.02	A	0.274	0.273* (CV: 1%)	28
	B	0.271
	C	0.275
2.08	A	0.271	0.255* (CV: 10%)	33
	B	0.227
	C	0.195
4.45	A	0.195	0.241* (CV: 17%)	36
	B	0.256
	C	0.273

 

^a Percent inhibition as compared to the pooled control was determined using the following
equation:

% inhibition = (((mean pooled control average specific growth rate) - (mean treatment average specific
growth rate))/ mean pooled control average specific growth rate) × 100

Key to Abbreviation: CV = coefficient of variance.

* Significant growth reduction as compared to the pooled control (Dunnett’s test, p < 0.05).

 

Validity criteria fulfilled:

yes

Conclusions:

The most sensitive EC50 is based on frond numbers and was determined to be 0.835 mg a.s./L.

Executive summary:

The most sensitive EC₅₀ is based on frond numbers and was determined to be 0.835 mg a.s./L.

Description of key information

The most sensitive EC₅₀ is based on frond numbers and was determined to be 0.835 mg a.s./L.

Key value for chemical safety assessment

EC50 for freshwater plants:

0.835 mg/L