Disodium [6-[(2-anilino-1-naphthyl)azo]-2,4-dinitrophenolato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]naphthalene-1-sulphonato(3-)]chromate(2-)

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:

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

Adequacy of study:

key study

Study period:

November from 09th to 20th, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

Details on the read-across are available in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Freshly prepared and old media were analysed. No additional replicates for the test item analysis were prepared.

Details on test solutions:

- Stock solution: 200 mg/l, freshly prepared with dilution water.
- Dispersion treatment: agitation.

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: duckweed.
- Source: Federal Environmental Agency (UBA), Schichauweg 58, 12307 Berlin, Germany.

CULTURE
- Culture: the species is bred at the test facility. Density is kept low to prevent conglomerates of plants on the surface. At least once per week, plants are transferred to freshly prepared growth medium. Growth media and breeding vessels are autoclaved before use to enable the breeding of axenic cultures.
- Culture vessels: crystallisation dishes, Vol. 900 ml, filled with ca. 500 ml growth medium, covered with glass lids.
- Medium: Swedish Standard (SIS) Medium, pH-value 6.5 : 0.2.
- Temperature: 24 ± 2 °C
- Light regime: continuous fluorescent light, 15 - 60 µE/m^2/s

ACCLIMATION
- Acclimation period: at least 7 days.
- Culturing media and conditions: acclimatization under test conditions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

22 - 23 °C

pH:

Control: 6.57 - 7.04
Vessels: 6.59 - 7.04

Nominal and measured concentrations:

2.00, 6.32, 20.2, 63.2, 200 mg/l, nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: crystallisation dishes with a 500 ml capacity.
- Type of cover: dishes were covered with glass lids and filled with 200 ml test solution.
- Agitation: yes
- No. of colonies per vessel: 4 uniform healthly-looking flants.
- No. of fronds per colony: 3 fronds each colony.
- Initial fron number: 12.
- No. of vessels per concentration: 3 replicates.
- No. of vessels per control: 6 replicates.

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: Swedish Standard (SIS) Medium according to the guideline.
- pH: 6.5 ± 0.2

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted adding 1 N NaOH.
- Photoperiod: continuous fluorescent light on the surface of the test medium. Difference of light intensity at any measured incubation place was less than 15 % from the mean value.
- Light intensity and quality: 6500 - 10000 Iux (corresponding to 85 - 135 µE/m^2/s)

EFFECT PARAMETERS MEASURED
Biological data
The amounts of plants were determined on start and end of the test and every 2 - 3 d. Every frond that were visibly projected beyond the edge of a parent frond were counted as a separate frond. Fronds that loose their pigmentation were not counted.
Observations of change in colour, break-up of plants and destructions of roots were made on every determination day and end of the test.
After 7 d determination of dry weight was carried out. Colonies from each test vessel were collected, rinsed with deionised water and then dried at 60 °C to a constant weight. Any root fragments were included. The starting biomass dry weight was determined based on a sample of colonies (same number of fronds as in the test vessels) taken from the same batch used to inoculate the test vessels.

Physicochemical data
pH-values were measured on start and end of the test.
The room temperature was measured and recorded continuously by a thermohygrograph.
Light intensity was determined before start of the test.

VALIDITY CRITERIA
The doubling time of frond number must be less than 2.5 d (60 h), corresponding to approximately an seven-fold increase in seven days and an average specific growth rate of 0.275/d.

RANGE-FINDING STUDY
- Test concentrations: 1, 10 and 100 mg/l.

Reference substance (positive control):

yes

Remarks:

3, 5-dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

103 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

22.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The test item was found to inhibit the growth of the monocotyledon Lemna minor after 7 days with the following effect values (nominal concentrations): the EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and dry weight inhibition of growth rate (ErdwC50) were 103 (75.1 - 137) mg/l and 73.8 (54.1 - 102) mg/l, respectively. The EC50-values with 95 % confidence intervals for inhibition of yield (EyC50) and dry weight inhibition of yield (EydwC50) was 22.8 (13.5 - 37.4) mg/l and 17.6 (12.5 - 22.1) mg/l.
The No Observed Effect Level effects observed was identified to be 6.32 mg/l, for both growth rate and yield and based on both frond number and dry weight.

MEASURED CONCENTRATION
The concentrations of test item were analysed at all concentration levels in fresh media and in old media after 7 days via HPLC-DAD analysis. The measured concentrations in the fresh media were in the range of 93 to 101 %. In the old media, measured concentrations were in the range of 79 to 102 % of the nominal values.

Results with reference substance (positive control):

The acute toxicity of 3,5-Dichlorophenol to the monocotyledon Lemna minor was determined over a period of 7 d, according to OECD guideline 221.
ErC50 (7d): 2.66 mg/l, based on frond number (95 % CI: 2.60 - 2.71 mg/l)
ErC50 (7d): 2.33 mg/l, based on dry weight (95 % CI: 2.07 - 2.61 mg/l)
EyC50 (7d): 2.14 mg/l, based on frond number (95 % CI: 2.06 - 2.23 mg/l)
EyC50 (7d): 1.64 mg/l, based on dry weight (95 % CI: 1.31 - 2.03 mg/l)

MAIN TEST

Frond number		Dry weight
Growth Rate inhibition (mg/l)
NOEC	6.32	NOEC	6.32
LOEC	20.0	LOEC	20.0
ErC10	13.1 (8.07 - 19.7)	ErdwC10	7.29 (4.37 - 11.6)
ErC20	25.2 (17.5 - 33.8)	ErdwC20	15.4 (10.4 - 21.2)
ErC50	103 (75.1 - 137)	ErdwC50	73.8 (54.1 - 102)
Yield inhibition (mg/l)
NOEC	6.32	NOEC	6.32
LOEC	20.0	LOEC	20.0
EyC10	12.0 (6.92 .- 17.0)	EydwC10	5.32 (<2.00 - 11.1)
EyC20	14.5 (8.29 - 18.2)	EydwC20	8.95 (5.71 - 13.4)
EyC50	22.8 (13.5 - 37.4)	EydwC50	17.6 (12.5 - 22.1)

Measure exposure concentrations

Nominal concentration (mg/l)	Start of exposure		End of exposure
	Meas. conc. (mg/l)	%	Meas. conc. (mg/l)	%
200	202	101	204	102
63.2	59.6	94	61.2	97
20.0	19.7	99	20.1	100
6.32	6.11	97	5.52	87
2.00	1.85	93	1.58	79
Control	< LOQm		< LOQm

Meas. conc: measured concentration of the test item, mean value of 2 injections (dilution factor taken into account)

%: percent of nominal concentration of the test item

LOQm: Limit of quantification of the analytical method (1.5 mg test item/l)

pH conditions

Nominal concentration (mg/l)	0d Start of exposure	7d End of exposure
200	6.61	6.72
63.2	6.59	6.84
20.0	6.61	6.99
6.32	6.61	7.03
2.00	6.71	7.04
Control	6.57	7.04

PRELIMINARY TEST RESULTS

Inhibition after 7 days

Nominal concentration (mg/l)	Specific growth rate inhibition (%)	Yield inhibition (%)
100	53	79
10	-1	-3
1	1	4

Validity criteria fulfilled:

yes

Remarks:

doubling times of the frond number in the definitive test was 2.11 days after 7 days of exposure in the control (required: < 2.5 days), corresponding to an average specific growth rate of 0.328 (10 -fold increase)

Conclusions:

ErC50 (7d): 103 mg/l, based on frond number.
ErC50 (7d): 73.8 mg/l, based on dry weight.

Executive summary:

The effects of test item on the growth of the monocotyledon species Lemna minor was determined according to the principles of OECD 221 (2006). A static exposure over 7 days was carried out. Based on a preliminary test, 5 nominal test item concentration levels were tested in a geometrical series with a dilution factor of √10: 2.00 - 6.32 - 20.0 - 63.2 - 200 mg/l. Three replicates were investigated for each test concentration and six for the control. Frond numbers were assessed on days 0, 3, 5 and 7. Environmental parameters light and temperature were within acceptable limits. Validity criteria of test guideline were fulfilled.

Concentrations of test item were analytically verified by HPLC-DAD: measured concentrations of test item at start of exposure (0 h) were in the range of 93 to 101 % of the nominal values; measured concentrations of test item at the end of exposure (7 d) were in the range of 79 to 102 % of nominal values, indicating that concentrations of test item were successfully maintained for test duration. The endpoints were assessed based on nominal concentrations of test item.

The 7-d ErC50, based on frond number, was determined to be 103 mg/l (75.1 - 137 mg/l); the 7-d ErC50, based on dry weight, was determined to be 73.8 mg/l (54.1 - 102 mg/l). The NOEC for growth rate inhibition was identified to be 6.32 mg/l, based on both frond number and dry weight.

The 7-d EyC50, based on frond number, was determined to be between 22.8 mg/l (13.5 - 37.4 mg/l). The 7-d EyC50, based on dry weight, was determined to be between 17.6 mg/l (12.5 - 22.1 mg/l). The NOEC for yield inhibition was identified to be 6.32 mg/l., based on both frond number and dry weight.

Conclusion

ErC50 (7d): 103 mg/l, based on frond number.

ErC50 (7d): 73.8 mg/l, based on dry weight.

Description of key information

ErC50 (7d): 103 mg/l, based on frond number.

ErC50 (7d): 73.8 mg/l, based on dry weight.

Key value for chemical safety assessment

EC50 for freshwater plants:

103 mg/L

Additional information

There are no information about the toxicity of Acid Black 222 to acquatic plants, thus the available information on the structural analogue Similar Substance 01 have been taken into consideration; the read across approach can be considered as appropriate and suitable to assess the property under investigation. Details are available in section 13.

The effects of Similar Substance 01 on the growth of Lemna minor were determined according to the principles of OECD guideline 221 (2006). The concentrations of test item were analytically verified by HPLC-DAD: measured concentrations of test item at start of exposure (0 h) were in the range of 93 to 101 % of nominal values; measured concentrations of test item at the end of exposure (7 d) were in the range of 79 to 102 % of nominal values, indicating that concentrations of test item were successfully maintained for the duration of the test. The endpoints were assessed based on nominal concentrations of test item.

The 7-d ErC50, based on frond number, was determined to be 103 mg/l (75.1 - 137 mg/l); the 7-d ErC50, based on dry weight, was determined to be 73.8 mg/l (54.1 - 102 mg/l). The NOEC for growth rate was identified to be 6.32 mg/l, based on both frond number and dry weight. The 7-d EyC50, based on frond number, was determined to be between 22.8 mg/l (13.5 - 37.4 mg/l). The 7-d EyC50, based on dry weight, was determined to be 17.6 mg/l (12.5 - 22.1 mg/l). The NOEC fo yield inhibition was identified to be 6.32 mg/l., based on both frond number and dry weight.