Disodium [4-hydroxy-3-[(2-hydroxy-4-nitrophenyl)azo]naphthalene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Remarks:

Lemna

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

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:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 20011/E TE
Physical state/ Appearance: Dark blue solid
Batch: 1309023 (China)
Purity: 65%
Expiry date: 30 September 2018
Storage conditions: Room temperature in the dark
Intended use/ Application: Unstandardised dyestuff

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation on Day 0 and from the pooled replicates on Day 7. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Remarks:

culture medium

Details on test solutions:

Range-finding test
A nominal amount of test item (154 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg ai/L test concentration from which a series of dilutions was made to give further test concentrations of 10, 1.0 and 0.10 mg ai/L.

Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
A nominal amount of test item (308 mg) was dissolved in culture medium and the volume adjusted to 2 liters to give a 100 mg ai/L test concentration from which a series of dilutions was made to give further test concentrations of 32, 10, 3.2 and 1.0 mg ai/L.

Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Lemna minor

Details on test organisms:

The test was carried out using Lemna minor. A culture of Lemna minor was obtained from Canadian Phycological Culture Centre, University of Waterloo, Ontario, Canada. Cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) for at least 7 days prior to the start of the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Test temperature:

24 ± 2 °C.

pH:

6.9 - 8.2

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

Range-finding Test: Nominal test concentrations of 0.10, 1, 10 and 100 mg ai/L.
Definitive Test: Nominal test concentrations of 1, 3.2, 10, 32 and 100 mg ai/L.

Details on test conditions:

The test was carried out using Lemna minor. A culture of Lemna minor was obtained from Canadian Phycological Culture Centre, University of Waterloo, Ontario, Canada. Cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) for at least 7 days

Range-finding Test
The test was conducted in glass conical flasks (250 mL). Two replicate flasks were prepared for each control and test concentration. The test item was dissolved directly in culture medium.
A nominal amount of test item (154 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg ai/L test concentration from which a series of dilutions was made to give further test concentrations of 10, 1.0 and 0.10 mg ai/L.
Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test the number of fronds present in each test and control culture was recorded along with observations on frond size, appearance, root length and number of colonies present. The flasks were then incubated at 24 ± 2 ºC under continuous illumination (intensity approximately 7000 lux) on a black, non-reflective surface for 7 days.
On Days 3 and 5 the test solutions were renewed, and observations on the test organisms were recorded on days 3, 5 and 7.
In order to determine the stability of the test item under test conditions a sample of each test concentration was taken for chemical analysis on Day 0 (fresh media) and Day 3 (old media). An additional sample of each test concentration was prepared on Day 0 and incubated alongside the test until Day 7 in order to determine stability over the entire test duration. All samples were stored frozen prior to analysis.
The results showed no significant effect on growth at the test concentrations of 0.10 and 1.0 mg ai/L. However, growth was observed to be reduced at 10 and 100 mg ai/L. Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg ai/L were selected for the definitive test.

Definitive Test
A nominal amount of test item (308 mg) was dissolved in culture medium and the volume adjusted to 2 liters to give a 100 mg ai/L test concentration from which a series of dilutions was made to give further test concentrations of 32, 10, 3.2 and 1.0 mg ai/L.
Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis on Day 0 and Day7.

Exposure Conditions
As in the range-finding test glass conical flasks were used. Three flasks each containing 250 mL of solution were prepared for the control and each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Each control and test flask was inoculated with 3 colonies of Lemna minor (total 9 fronds). The flasks were then incubated at 24 ± 2 ºC under constant illumination (intensity approximately 7000 lux) on a black, non-reflective surface for 7 days.
A static testing regime was employed.

Test Organism Observations
The number of fronds present in each test and control culture was recorded on days 0, 3, 5 and 7 along with observations on frond size, appearance, root length and number of colonies present.

In addition, the dry weight of the fronds in each control and treatment group was determined on day 7. At the start of the test six replicate samples of fronds identical to those used to inoculate the test vessels were taken and the dry weight determined. At the end of the test the dry weight of colonies from each control and test vessel was determined by blotting the colonies dry and drying at 60 °C to constant weight. prior to the start of the test.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

82 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

50 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Dry weight Average specific growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

2.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Dry weight Yield

Details on results:

Range-finding Test
The results showed no significant effect on growth at the test concentrations of 0.10 and 1.0 mg ai/L. However, growth was observed to be reduced at 10 and 100 mg ai/L.

Chemical analysis of the test preparations on Day 0 showed measured test concentrations to range from 78% to 94% of nominal. A slight decline in measured test concentrations was observed on Day 3 in the range of 68 % to 94 % of nominal, and on Day 7 in the range of 70 % to 89 % of nominal.

Growth Data Based on Frond Number
Accordingly, the following results based on inhibition of average specific growth rate and yield were determined from the frond number data:

Average Specific Growth Rate
ErC10 (frond number) = 3.5 mg ai/L
ErC20 (frond number) = 11 mg ai/L
ErC50 (frond number) = 82 mg ai/L
Where: ErCx = the test concentration that reduced average specific growth rate by x%.
Statistical analysis of the average specific growth rate data was carried out for the control and all test concentrations using one-way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control and 0.64 mg ai/L test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of average specific growth rates calculated from frond numbers was 0.64 mg ai/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 2.3 mg ai/L.


Yield
EyC10 (frond number) = 0.63 mg ai/L
EyC20 (frond number) = 2.1 mg ai/L
EyC50 (frond number) = 17 mg ai/L
Where: EyCx = the test concentration that reduced yield by x %.
Statistical analysis of the yield data was carried out for the control and all test concentrations using one-way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control and 0.64 mg ai/L test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from frond numbers was 0.64 mg ai/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 2.3 mg ai/L.


Growth Data Based on Dry Weight
Accordingly, the following results based on inhibition of average specific growth rate and yield were determined from the dry weight data:
Average Specific Growth Rate
ErC10 (dry weight) = 1.8 mg ai/L
ErC20 (dry weight) = 6.1 mg ai/L
ErC50 (dry weight) = 50 mg ai/L
Where: ErCx = the test concentration that reduced average specific growth rate by x %.
Statistical analysis of the average specific growth rate data was carried out for the control and all test concentrations using one-way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control and 0.64 mg ai/L test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of average specific growth rate calculated from dry weight was 0.64 mg ai/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 2.3 mg ai/L.


Yield
EyC10 (dry weight) = 0.58 mg ai/L
EyC20 (dry weight) = 1.8 mg ai/L
EyC50 (dry weight) = 13 mg ai/L
Where: EyCx = the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out for the control and all test concentrations using one-way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control and 0.64 mg ai/L test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from dry weight was 0.64 mg ai/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 2.3 mg ai/L.

Results with reference substance (positive control):

A positive control (Harlan Study Number 41403075) used 3,5-dichlorophenol as the reference item at concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L.

Exposure conditions and data evaluation for the positive control were like those in the definitive test.

Exposure of Lemna minor to the reference item gave the following results:
The average specific growth rate (frond number) EC50 was 3.4 mg/L with 95 % confidence limits of 3.1 - 3.6 mg/L. The NOEC was 2.5 mg/L and the LOEC was 5.0 mg/L.
The average specific growth rate (dry weight) EC50 was 3.4 mg/L with 95 % confidence limits of 3.2 - 3.7 mg/L. The NOEC was 1.25 mg/L and the LOEC was 2.5 mg/L.
The yield (frond number) EC50 was 2.6 mg/L with 95 % confidence limits of 2.4 - 2.9 mg/L. The NOEC was 2.5 mg/L and the LOEC was 5.0 mg/L.
The yield (dry weight) EC50 was 2.7 mg/L with 95 % confidence limits of 2.5 - 2.9 mg/L. The NOEC was 1.25 mg/L and the LOEC was 2.5 mg/L.

The results from the positive control with 3,5-dichlorophenol were within the normal ranges for this reference item.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981), and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955), were carried out on the average specific growth rate and yield data at 7 days for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999-2001).

Verification of Test Concentrations

Chemical analysis of the test preparations on Day 0 (fresh media) showed measured test concentrations to range from 0.90 to 95 mg ai/L. A slight decline in measured test concentration was observed on Day 7 in the range of 0.46 to 88 mg ai/L.

 

Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC₅₀ values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be:

 

Nominal Test Concentration (mg ai/L)	Geometric Mean Measured Test Concentration (mg ai/L)	Expressed as a % of the Nominal Test Concentration
1.0	0.64	64
3.2	2.3	72
10	9.1	91
32	30	94
100	91	91

Validation Criteria

The following data show that the doubling time of the control cultures was 1.76 days in line with the OECD Guideline that states the doubling time should be less than 2.5 days:

 

Mean frond number in control cultures at day 0: 9

Mean frond number in control cultures at day 7: 99

Water Quality Criteria

Temperature was maintained at 24 ± 2 ºC throughout the test.

Frond Numbers and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration (mg ai/L)		Number of Fronds				Average Specific Growth Rate		Yield
		Day 0	Day 3	Day 5	Day 7	(0 – 7 Day)	% Inhibition	(0 – 7 Day)	% Inhibition
Control	R1	9	24	49	112	0.360	-	103	-
	R2	9	29	65	146	0.398		137
	Mean	9	27	57	129	0.379		120
0.10	R1	9	30	66	138	0.390	[3]	129	[9]
	R2	9	27	62	141	0.393		132
	Mean	9	29	64	140	0.392		131
1.0	R1	9	28	62	132	0.384	0	123	2
	R2	9	28	63	121	0.371		112
	Mean	9	28	63	127	0.378		118
10	R1	9	24	39	87	0.324	17	78	39
	R2	9	24	40	76	0.305		67
	Mean	9	24	40	82	0.315		73
100	R1	9	21	23	43	0.223	38	34	68
	R2	9	23	32	50	0.245		41
	Mean	9	22	28	47	0.234		38

R₁– R₂= Replicates 1 and 2

Frond Numbers from the Definitive Test

Nominal Concentration (mg ai/L)		Number of Fronds
		Day 0	Day 3	Day 5	Day 7
Control	R1	9	23	51	113
	R2	9	24	49	93
	R3	9	23	46	92
	Mean	9	23	49	99
1.0	R1	9	21	38	89
	R2	9	19	42	96
	R3	9	20	41	99
	Mean	9	20	40	95
3.2	R1	9	20	37	70
	R2	9	21	36	67
	R3	9	20	40	82
	Mean	9	20	38	73
10	R1	9	21	33	63
	R2	9	19	35	64
	R3	9	22	36	65
	Mean	9	21	35	64
32	R1	9	20	34	48
	R2	9	21	31	49
	R3	9	20	32	55
	Mean	9	20	32	51
100	R1	9	14	22	25
	R2	9	21	25	29
	R3	9	14	17	25
	Mean	9	16	21	26

R₁– R₃= Replicates 1 to 3

Inhibition of Average Specific Growth Rate and Yield Based on Frond Numbers

Nominal Concentration (mg ai/L)		Average Specific Growth Rate		Yield
		(0 – 7 Day)	% Inhibition	(0 – 7 Day)	% Inhibition
Control	Mean	0.342	-	90	-
	SD	0.016		12
1.0	Mean	0.336	2	86	4
	SD	0.008		5
3.2	Mean	0.299	13	64	29
	SD	0.015		8
10	Mean	0.280	18	55	39
	SD	0.002		1
32	Mean	0.247	28	42	53
	SD	0.011		4
100	Mean	0.153	55	17	81
	SD	0.012		2

SD– Standard Deviation

Dry Weights from the Definitive Test

Nominal Concentration (mg ai/L)		Dry Weight (mg)
		Day 0*	Day 7
Control	R1		8.6
	R2		8.4
	R3		8.1
	Mean	1.2	8.4
1.0	R1		8.2
	R2		8.5
	R3		8.3
	Mean	1.2	8.3
3.2	R1		5.6
	R2		6.0
	R3		7.2
	Mean	1.2	6.3
10	R1		5.1
	R2		4.7
	R3		5.1
	Mean	1.2	5.0
32	R1		4.2
	R2		3.6
	R3		4.3
	Mean	1.2	4.0
100	R1		2.3
	R2		3.1
	R3		2.4
	Mean	1.2	2.6

*Mean value determined from 6 replicate weighings

R₁– R₃= Replicates 1 to 3

Inhibition of Average Specific Growth Rate and Yield Based on Dry Weight

Nominal Concentration (mg ai/L)		Average Specific Growth Rate		Yield
		(0 – 7 Day)	% Inhibition	(0 – 7 Day)	% Inhibition
Control	Mean	0.277	-	7.2	-
	SD	0.004		0.3
1.0	Mean	0.277	0	7.1	1
	SD	0.003		0.2
3.2	Mean	0.235	15	5.1	29
	SD	0.019		0.8
10	Mean	0.203	27	3.8	47
	SD	0.007		0.2
32	Mean	0.173	38	2.8	61
	SD	0.014		0.4
100	Mean	0.109	61	1.4	81
	SD	0.023		0.4

SD– Standard Deviation

pH Values in the Definitive Test

Nominal Concentration (mg ai/L)		Time (Days)
		0	7
Control	R1	6.9	8.2
	R2	6.9	8.1
	R3	6.9	8.0
1.0	R1	6.8	7.9
	R2	6.8	7.8
	R3	6.8	7.8
3.2	R1	6.8	7.6
	R2	6.8	7.5
	R3	6.8	7.5
10	R1	6.8	7.5
	R2	6.9	7.5
	R3	6.9	7.5
32	R1	6.9	7.4
	R2	7.0	7.4
	R3	7.0	7.4
100	R1	7.0	7.4
	R2	7.0	7.4
	R3	7.0	7.3

R₁– R₃= Replicates 1 to 3

Exposure of Lemna minor to the test item based on the geometric mean measured test concentrations gave the following results:

 

Response Variable	Measurement Variable	EC50 (mg ai/L)	No Observed Effect Concentration (NOEC) (mg ai/L)	Lowest Observed Effect Concentration (LOEC) (mg ai/L)
Average Specific Growth Rate	Frond Number	82	0.64	2.3
	Dry Weight	50	0.64	2.3
Yield	Frond Number	17	0.64	2.3
	Dry Weight	13	0.64	2.3

 

Validity criteria fulfilled:

yes

Conclusions:

The average specific growth rate (frond number) EC50 was 82 mg ai/L, while the NOEC was 0.64 mg/L.

Executive summary:

A study was performed to assess the effect of the test item on the growth of the freshwater plant Lemna minor. The method followed that described in the OECD Guideline No. 221 “Lemna Growth Inhibition Test (March 2006)”.

 

Following a preliminary range-finding test, Lemna minor was exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg active ingredient (ai)/L (three replicate flasks per concentration) for a period of 7 days, under constant illumination at a temperature of 24 ± 2 °C. 

The number of fronds in each control and treatment group was recorded on days 0, 3, 5 and 7 along with observations on plant development.

Chemical analysis of the test preparations on Day 0 (fresh media) showed measured test concentrations to range from 0.90 to 95 mg ai/L. A slight decline in measured test concentration was observed on Day 7 in the range of 0.46 to 88 mg ai/L.

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data.

Exposure of Lemna minor to the test item based on the geometric mean measured test concentrations gave the following results:

The average specific growth rate (frond number) EC50 was 82 mg ai/L.

The average specific growth rate (dry weight) EC50 was 50 mg ai/L.

The yield (frond number) EC50 was 17 mg ai/L.

The yield (dry weight) EC50 was 13 mg ai/L.

The NOEC was 0.64 mg/L and the LOEC was 2.3 mg ai/L.

Description of key information

The effect of the test item on the growth of Lemna minor has been investigated over a 7-day period and gave the following results based on the geometric mean measured test concentrations:
The average specific growth rate (frond number) EC50 was 82 mg ai/L.
The average specific growth rate (dry weight) EC50 was 50 mg ai/L.
The yield (frond number) EC50 was 17 mg ai/L.
The yield (dry weight) EC50 was 13 mg ai/L.
The NOEC was 0.64 mg/L and the LOEC was 2.3 mg ai/L.

Key value for chemical safety assessment

EC50 for freshwater plants:

82 mg/L

EC10 or NOEC for freshwater plants:

0.64 mg/L