Hydrogen bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

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:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From June 25 to July 8, 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The complete read across justification is detailed in section 13. Source study has reliability 1.

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

yes

Remarks:

pH increased by more than 1.5 units during the test at several time points; howvere, test is still valid, as it was shown that validity criteria are met.

GLP compliance:

yes (incl. QA statement)

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:

- Test organism: aquatic plant duckweed, Lemna gibba G3 (Alismatales: Araceae).
- Source: aseptic clones from the cultivation of the testing facility, 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.
- Culturing media and conditions: 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 at least 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

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

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 these 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, EC20 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:

endpoint	test item [mg/l] nominal
	EC10	EC20	EC50
yield of frond number - Probit analysis (logistic distribution)	0.372	0.804	3.00
growth rate of frond number - Probit analysis (Gompertz distribution)	1.05	2.93	13.8
yield of dry weight - Probit distribution (normal distribution)	0.737	1.33	4.09
growth rate of dry weight - Probit analysis (Gompertz distribution)	1.38	3.20	11.4

endpoint	test item [mg/l] nominal
	NOEC Dunnets-t-test	LOEC Dunnetts-t-test
yield of frond number	0.610	1.95
growth rate of frond number	0.610	1.95
yield of dry weight	0.610	1.95
growth rate of dry weight	0.610	1.95

Validity criteria fulfilled:

yes

Conclusions:

Growth rate of frond numbers based on Probit analysis (Gompertz distribution): EC50 (7d) = 13.8 mg/l (nominal) and 12.3 mg/l (measured).
Growth rate of dry weight based on Probit analysis (Gompertz distribution): EC50 (7d)= 11.4 mg/l (nominal) and 10.1 mg/l (measured).

Executive summary:

Method

Inhibitory effects of test substance to the duckweed Lemna minor were investigated over a period of 7 days under semi-static exposure conditions, based on the frond number and biomass (dry weight) following OECD guideline 221.

Test solutions were prepared at nominal concentrations of 20, 6.25, 1.95, 0.610, 0.191 mg/l, corresponding to 18.4, 5.25, 1.64, 0.397 and 0.085 mg/l of measured concentrations. The lowest concentration was below the LOQ of the analytical method and set to LOQ/2.

Test concentrations were determined at day 0, 2 (aged and fresh solutions) and 5 (aged and fresh solutions). Effective concentrations (E_rC50 and E_yC50) were assessed based on the geometric mean of the measured concentrations. Measured concentrations were in the range of 45 to 92 % of nominal.

Frond number and biomass (dry weight) were assessed as growth rate and yield.

Results

The initial mean measured concentration of 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 measured concentrations were reported as geometric mean of concentrations measured at different time points for each concentration level.

endpoint	test item [mg/l] nominal			test item [mg/l] measured
	EC10	EC20	EC50	EC10	EC20	EC50
yield of frond number - Probit analysis (logistic distribution)	0.372	0.804	3.00	0.297	0.656	2.54
growth rate of frond number - Probit analysis (Gompertz distribution)	1.05	2.93	13.8	0.837	2.44	12.3
yield of dry weight - Probit distribution (normal distribution)	0.737	1.33	4.09	0.591	1.09	3.48
growth rate of dry weight - Probit analysis (Gompertz distribution)	1.38	3.20	11.4	1.10	2.67	10.1

endpoint	test item [mg/l] nominal		test item [mg/l] measured
	NOEC Dunnets-t-test	LOEC Dunnetts-t-test	NOEC Dunnets-t-test	LOEC Dunnetts-t-test
yield of frond number	0.610	1.95	0.397	1.64
growth rate of frond number	0.610	1.95	0.397	1.64
yield of dry weight	0.610	1.95	0.397	1.64
growth rate of dry weight	0.610	1.95	0.397	1.64

Description of key information

E_rC50 (7d) = 12.3 mg/l based on Probit analysis (Gompertz distribution) for frond number

E_rC50 (7d) = 10.1 mg/l based on Probit analysis (Gompertz distribution) for dry weight

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

No data on toxicity of Solvent Orange 062 to aquatic plants was available, thus the assessment relied on a read across approach. Solvent Orange 062 and Similar Substance 01 are chromium complexes with 2 ligands. In both cases, the 2 ligands have the same chemical structure, i.e. a phenyl ring connected via diazo bond to a phenyl pyrazole. These ligands show minor differences in the position of substituents, i.e. hydroxy and nitro gorups, as well as in the presence of a sulfonate group in Similar Substance 01.

All information about the read across process is inserted in section 13.

In the available study conducted according to OECD guideline 221, significant inhibitory effects were determined for all parameters at 3 highest measured concentrations of 1.64, 5.25 and 18.4 mg/l. Upon a 7 -day exposure, effect levels for growth rate (E_rCx) and yield (E_yCx) of frond number and dry weight were estimated. Following results were obtained:

- as for frond number, actual E_yC10 = 0.297 mg/l and actual E_rC10 = 0.837 mg/l

- as for dry weight, actual E_yC10 = 0.591 mg/l and actual E_rC10 = 1.10 mg/l

- as for frond number, actual E_yC50 = 2.54 mg/l and actual E_rC50 = 12.3 mg/l

- as for dry weigth, actual E_yC50 = 3.48 mg/l and actual E_rC50 = 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).