Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

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:

25 Jun 2014 – 08 Jul 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

yes

Remarks:

No major (the pH increased by more than 1.5 units during the test at several time points. The test is still valid, as it was shown that validity criteria are met).

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

None

Analytical monitoring:

yes

Details on sampling:

After sampling, the test medium samples (10 mL) were stored deep-frozen (below 18 °C) until analysis.

Vehicle:

no

Details on test solutions:

The necessary amount of test item for preparing the stock solution S1 was weighed and transferred to a volumetric flask. Test medium was added up to the bench mark and the solution was homogenised by shaking. Lower concentrations of the test item were prepared by dilution of the stock solution.

Test organisms (species):

Lemna gibba

Details on test organisms:

The test organism was the aquatic plant duckweed, Lemna gibba G3 (Alismatales: Araceae). The plants used in this study were taken as aseptic clones from the cultivation of the testing facility. They were primarily cultured by Dr. Janet Slovin, Horticulture Crops Quality Laboratory, U.S. Department of Agriculture. BARC-West, Bldg. 050 HH-4, Beltsville, MD 20705, U.S.A. The plants were cultured in appropriate glass beakers under continuous illumination with OSRAM light tubes (L30/32-930 and L30/W72-965), at 6500 - 7000 lux. The temperature was 24 ± 2 °C. Before the test was started, plants were cultured under the same illumination and temperature conditions as those to be used for the toxicity test. Young, light-green plants of similar size and comprising 2 - 5 fronds were transferred onto fresh medium and cultured for ≥3 weeks prior to testing, with two further transfers onto fresh medium before initiating the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

23.1 – 24.6 °C

Nominal and measured concentrations:

see table below

Details on test conditions:

Test procedure: semi-static test
Duration: 7 days
Temperature: 23.1 – 24.6 °C
pH-value of fresh solution: 7.45 – 7.65
Light intensity (mean): ~ 6800 lux
Light tubes: OSRAM light tubes (L 30 W77 and L 30 W25)
Exposure to light: continuously
Test vessels: 250 mL glass beakers filled with 150 mL
Number of fronds: 12 per test vessel

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.297 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield of frond number

Remarks on result:

other: 0.0997-0.554

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

0.656 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield of frond number

Remarks on result:

other: 0.297-1.04

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

2.54 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield of frond number

Remarks on result:

other: 1.80-3.30

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.397 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield of frond number

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

1.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield of frond number

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.837 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate of frond number

Remarks on result:

other: 0.280-1.53

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

2.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate of frond number

Remarks on result:

other: 1.27-3.60

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

12.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate of frond number

Remarks on result:

other: 9.34-17.5

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.37 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate of frond number

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

1.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate of frond number

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.591 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield of dry weight

Remarks on result:

other: 0.300-0.916

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

1.09 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield of dry weight

Remarks on result:

other: 0.653-1.52

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

3.48 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield of dry weight

Remarks on result:

other: 2.71-4.32

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.397 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield of dry weight

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

1.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield of dry weight

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate of dry weight

Remarks on result:

other: 0.506-1.78

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

2.67 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate of dry weight

Remarks on result:

other: 1.62-3.70

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

10.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate of dry weight

Remarks on result:

other: 8.09-12.9

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.397 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate of dry weight

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

1.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate of dry weight

Reported statistics and error estimates:

The statistical evaluation for day 7 was performed for yield of frond numbers, growth rate of frond numbers, growth rate dry weight and yield of dry weight. For the determination of endpoints based on actual concentrations the two lowest test item concentrations of 0.0186 and 0.0596 mg/L were not considered, as theses concentration levels were not analysed. A test for normality of the data was performed by calculating the Shapiro-Wilk’s statistic, a test for homogeneity of the data was performed according to Levene. The NOEC and LOEC were determined by using a multiple comparison method (Dunnett`s t-test). The EC10, 20- and EC50-values for yield of frond number, growth rate of frond number, yield of dry weight and growth rate of dry weight were determined by probit analysis following normal, logistic or Gompertz procedure. The evaluation of data was performed by SAS® (2002-2010).

Toxicological endpoints based on nominal concentrations:

	Test item [mg/L] nominal
Endpoint	EC10	EC20	EC50	NOEC^4)	LOEC^4)
Yield of frond numbers	0.372^2)	0.804^2)	3.00^2)	0.610 0.610 0.610 0.610	1.95
Growth rate of frond numbers	1.05^3)	2.93^3)	13.8^3)		1.95
Yield of dry weight	0.737^1)	1.33^1)	4.09^1)		1.95
Growth rate of dry weight	1.38^3)	3.20^3)	11.4^3)		1.95

1) Probit analysis following normal distribution

2) Probit analysis following logistic distribution

3) Probit analysis following Gompertz distribution

4) following Dunnetts-t-test

* based on the geometric mean of the analysed concentration levels

Validity criteria fulfilled:

yes

Conclusions:

The nominal EC50-value for yield of frond numbers was determined to be 3.00 mg/L, the nominal EC50-value for growth rate of frond numbers was determined to be 13.8 mg/L. The nominal EC50-value for yield of dry weight was determined to be 4.09 mg/L, the nominal EC50-value for growth rate of dry weight was determined to be 11.4 mg/L. The actual EC50-value for yield of frond numbers was determined to be 2.54 mg/L, the actual EC50-value for growth rate of frond numbers was determined to be 12.3 mg/L. The actual EC50-value for yield of dry weight was determined to be 3.48 mg/L, the actual EC50-value for growth rate of dry weight was determined to be 10.1 mg/L.

Executive summary:

Toxicity of FAT 20060/F to aquatic plant Lemna major was evaluated in a study conducted according to OECD Guideline 221. The test concentrations used were 0.191, 0.610, 1.95, 6.25 and 20.0 mg/L. The initial mean measured concentration of FAT 20060/F in the test item solutions was 77 % of nominal. The overall mean concentration of fresh and aged samples was 72 % of nominal. Toxicological endpoints were evaluated using nominal concentrations of the test item and actual concentrations based on the geometric mean concentration of each test item concentration level.

	Test item [mg/L] nominal
Endpoint	EC10	EC20	EC50	NOEC^4)	LOEC^4)
Yield of frond numbers	0.372^2)	0.804^2)	3.00^2)	0.610 0.610 0.610 0.610	1.95
Growth rate of frond numbers	1.05^3)	2.93^3)	13.8^3)		1.95
Yield of dry weight	0.737^1)	1.33^1)	4.09^1)		1.95
Growth rate of dry weight	1.38^3)	3.20^3)	11.4^3)		1.95
	Test item [mg/L] actual*
Endpoint	EC10	EC20	EC50	NOEC^4)	LOEC^4)
Yield of frond numbers	0.297^2)	0.656^2)	2.54^2)	0.397 0.397 0.397 0.397	1.64
Growth rate of frond numbers	0.837^3)	2.44^3)	12.3^3)		1.64
Yield of dry weight	0.591^1)	1.09^1)	3.48^1)		1.64
Growth rate of dry weight	1.10^3)	2.67^3)	10.1^3)		1.64

1) Probit analysis following normal distribution

2) Probit analysis following logistic distribution

3) Probit analysis following Gompertz distribution

4) following Dunnetts-t-test

* based on the geometric mean of the analysed concentration levels

 

Significant inhibitory effects were determined for all parameters at 1.95, 6.25 and 20.0 mg/L (nominal) and 1.64, 5.25 and 18.4 mg/L (actual). The overall LOEC was therefore determined to be 1.95 mg/L (nominal) and 1.64 mg/L (actual), the corresponding NOEC at 0.610 mg/L (nominal) and 0.397 mg/L (actual). The nominal EC10-value for yield of frond numbers was determined to be 0.372 mg/L, the nominal EC10-value for growth rate of frond numbers was determined to be 1.05 mg/L. The nominal EC10-value for yield of dry weight was determined to be 0.737 mg/L, the nominal EC10-value for growth rate of dry weight was determined to be 1.38 mg/L. The actual EC10-value for yield of frond numbers was determined to be 0.297 mg/L, the actual EC10-value for growth rate of frond numbers was determined to be 0.837 mg/L. The actual EC10-value for yield of dry weight was determined to be 0.591 mg/L, the actual EC10-value for growth rate of dry weight was determined to be 1.10 mg/L. The nominal EC20-value for yield of frond numbers was determined to be 0.804 mg/L, the nominal EC20-value for growth rate of frond numbers was determined to be 2.93 mg/L. The nominal EC20-value for yield of dry weight was determined to be 1.33 mg/L, the nominal EC20-value for growth rate of dry weight was determined to be 3.20 mg/L. The actual EC20-value for yield of frond numbers was determined to be 0.656 mg/L, the actual EC20-value for growth rate of frond numbers was determined to be 2.44 mg/L. The actual EC20-value for yield of dry weight was determined to be 1.09 mg/L, the actual EC20-value for growth rate of dry weight was determined to be 2.67 mg/L. The nominal EC50-value for yield of frond numbers was determined to be 3.00 mg/L, the nominal EC50-value for growth rate of frond numbers was determined to be 13.8 mg/L. The nominal EC50-value for yield of dry weight was determined to be 4.09 mg/L, the nominal EC50-value for growth rate of dry weight was determined to be 11.4 mg/L. The actual EC50-value for yield of frond numbers was determined to be 2.54 mg/L, the actual EC50-value for growth rate of frond numbers was determined to be 12.3 mg/L. The actual EC50-value for yield of dry weight was determined to be 3.48 mg/L, the actual EC50-value for growth rate of dry weight was determined to be 10.1 mg/L. The test is valid as the doubling time of control frond numbers was calculated to be 37.9 h, corresponding to 1.581 days at day 7. This is less than the maximum allowable doubling time set in OECD guideline 221 (60 h, 2.5 days).

Description of key information

The actual EC50 value for growth rate of frond numbers was determined to be 12.3 mg/L.

Key value for chemical safety assessment

EC50 for freshwater plants:

12.3 mg/L

EC10 or NOEC for freshwater plants:

0.837 mg/L

Additional information

In the study evlauting toxicity to Lemna gibba, significant inhibitory effects were determined for all parameters at 1.95, 6.25 and 20.0 mg/L (nominal) and 1.64, 5.25 and 18.4 mg/L (actual). The actual EC10-value for yield of frond numbers was determined to be 0.297 mg/L, the actual EC10-value for growth rate of frond numbers was determined to be 0.837 mg/L. The actual EC10-value for yield of dry weight was determined to be 0.591 mg/L, the actual EC10-value for growth rate of dry weight was determined to be 1.10 mg/L. The actual EC50-value for yield of frond numbers was determined to be 2.54 mg/L, the actual EC50-value for growth rate of frond numbers was determined to be 12.3 mg/L. The actual EC50-value for yield of dry weight was determined to be 3.48 mg/L, the actual EC50-value for growth rate of dry weight was determined to be 10.1 mg/L. The test is valid as the doubling time of control frond numbers was calculated to be 37.9 h, corresponding to 1.581 days at day 7. This is less than the maximum allowable doubling time set in OECD guideline 221 (60 h, 2.5 days).