Trisodium bis[3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]naphthalene-1-sulphonato(3-)]chromate(3-)

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:

From July 18 August 21, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to other study

Remarks:

validated analytical method

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

adopted on 23 March 2006

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Concentrations were determined at the beginning and at the end of the test period. During the range finding study and main study, analytical samples (plus retain samples) were taken from a randomly chosen replicate per treatment group at beginning and at the end of the test (i.e. at 0 hour and 7 days). About 10 ml of sample was collected from all test concentration in duplicates.

Vehicle:

no

Details on test solutions:

Stock solution was freshly prepared in the Swedish Standard (SIS) growth medium. Required quantity of test item was taken in a beaker, in which small volume of Swedish Standard (SIS) growth medium was added; the solution was stirred well using glass rod. After complete solubility, test formulation was transferred to measuring cylinder, beaker was rinsed with Swedish Standard (SIS) growth medium and transferred again to measuring cylinder; the rinsing procedure was repeated until the complete transfer of test contents. Finally, the volume made up to required volume using Swedish Standard (SIS) growth medium.
A homogeneous distribution of the test item within the stock solution was achieved by stirring on magnetic stirrer before preparation of the test medium.
The test concentrations were prepared by adding appropriate amount of stock solutions and made up to required volume using Swedish Standard (SIS) growth medium.

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Source: in house maintained culture of Lemna minor, obtained from the Department of Botany, Tumkur University. Lemna minor culture is periodically sub cultured and maintained with the illumination and temperature of 6500 to 10000 lux and 22 to 26°C respectively. Lemna minor was maintained separately on Steinberg medium and Swedish Standard (SIS) media.

ADAPTATION
- Pre-culturing period: the pre culture was initiated 7 days before start of the test.
- Culturing media and conditions: 10 beakers containing 100 ml of culture medium; pre cultures are incubated under same conditions as the test cultures.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

23.9 - 24.5 °C

pH:

6.39 - 6.54

Nominal and measured concentrations:

0.9 , 2, 4.2, 9.4, 20.6, 45 and 100 mg dyestuff/l

Details on test conditions:

TEST SYSTEM
- Test vessel: sterilized, glass beakers of 150 ml capacity.
- Fill volume: the volume of the test solution at the initiation of the test in the test vessel was 100 ml.
- Cover: the test vessels were covered, allowing necessary air exchange, to minimise accidental contamination.
- No. of colonies per vessel: the initial Lemna minor colonies comprising 3 fronds.
- No. of fronds per colony: the initial number of frond number in all treatment replicates on day 0 was 12.
- No. of vessels per concentration: three replicates.
- No. of vessels per control: six replicates.

GROWTH MEDIUM
- Standard medium used: yes, Standard (SIS) growth medium.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: standard (SIS) growth medium, prepared in accordance with OECD guideline 221.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination.
- Light intensity and quality: 6854 to 6932 lux, with white fluorescent lamps.

EFFECT PARAMETERS MEASURED
- Counting of frond number: fronds number at the start on day 0, day 4 and on day 7 was counted in each test vessel and recorded.
- Observation of fronds: fronds number appearing normal or abnormal was determined at beginning of test and on day 3, day 6 and at end of the test. Changes in plant development were recorded. Changes in plant development (e.g. frond size, appearance, indication of necrosis, chlorosis or gibbosity, colony break-up or loss of buoyancy and in root length and appearance) was recorded.
- Measurement of dry weight: dry weight was determined at test start from representative sample of the inoculum culture. Consistent with the approach taken in the test. At the end of the test, the plant material from each replicate of test concentration and control vessel was used to determine dry weight. To determine the dry weight they are blotted to remove excess water and then dried at 60 °C to a constant weight. Root fragments were included and dry weight was measured.

ENVIRONMENTAL PARAMETERS MEASUREMENT
The temperature (culture medium) and light intensity (at points the same distance from the light source as the Lemna fronds) was monitored once in a day during pre-culture and exposure during range finding study and main study. pH values of test concentrations (fresh and spent) were measured on day 0 and on day 7 at the termination of the test during range finding and main study.

RANGE-FINDING STUDY
- Test concentrations: 0.01, 0.1, 1, 10, 50 and 100 mg dyestuff/l.
- No. of colonies per vessel: initial Lemna minor colonies comprising 3 fronds.
- No. of fronds per colony: the initial number of frond number in all treatment replicates on day 0 was 9.
- No. of vessels per concentration: two replicates.
- No. of vessels per control: four replicates.
- Test condition: the temperature was found to be 24.1 to 24.8 °C and light intensity of 6840 to 6919 lux during pre-culture and exposure; the pH of test concentrations was found to be 6.43 to 6.51.
- Results used to determine conditions for the definitive study: the percentage of inhibition in growth rate resulted to be 0.0, 4.4, 13.2, 24.0, 62.3 and 100 based on the frond number at the concentrations of 0.01, 0.1, 1, 10, 50 and 100 mg dyestuff/l, nominal; the percentage of inhibition in yield resulted to be 0.9, 10.6, 27.4, 44.2, 82.3 and 100 based on the frond number at the concentrations of 0.01, 0.1, 1, 10, 50 and 100 mg dyestuff/l, nominal.

VALIDITY CRITERIA OF THE TEST
For the test to be valid, the doubling time of frond number in the control must be less than 2.5 days (60 h), corresponding to approximately a seven-fold increase in seven days and an average specific growth rate of 0.275 d-1.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

27 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

MAIN TEST
ErC50 (7d): 27 mg dyestuff /l (nominal) (95 % CL: 15.99 - 45.34 mg dyestuff/l), based on frond number
EyC50 (7d): 10 mg dyestuff /l (nominal) (95 % CL: 6.94 - 14.54 mg dyestuff/l), based on frond number
ErC50 (7d): 32 mg dyestuff /l (nominal) (95 % CL: 23.23 – 44.01 mg dyestuff/l), based on biomass (dry weight)
EyC50 (7d): 11 mg dyestuff/l (nominal) (95 % CL: 8.86 - 13.20 mg dyestuff/l), based on biomass (dry weight)

NOErC (7d): 2 mg dyestuff/l (nominal), based on frond number
NOEyC (7d): 0.9 mg dyestuff/l (nominal), based on biomass (dry weight)
LOErC (7d): 4.2 mg dyestuff/l (nominal), based on frond number
LOEyC (7d): 2 mg dyestuff/l (nominal), based on biomass (dry weight)

Percent inhibition in growth rate:
- for frond number, it was found to be 3.9, 7.0, 19.0, 24.4, 30.7, 72.8 and 100 % at nominal test concentrations of 0.9, 2, 4.2, 9.4, 20.6, 45 and 100 mg dyestuff/l;
- for biomass (dry weight), it was found to be 1.4, 7.7, 15.7, 25.4, 40.5, 54.1 and 100 % at nominal tested concentrations of 0.9, 2.0, 4.2, 9.4, 20.6, 45 and 100 mg dyestuff/l.

Percent inhibition in yield:
- for frond number, it was found to be 8.7, 14.8, 36.2, 44.2, 52.8, 88.4 and 100 % at nominal test concentrations 0.9, 2, 4.2, 9.4, 20.6, 45 and 100 mg dyestuff/l;
- for biomass (dry weight), it was found to be 3.7, 17.7, 32.9, 48.1, 66.2, 78.2 and 100 % at nominal tested concentrations of 0.9, 2.0, 4.2, 9.4, 20.6, 45 and 100 mg dyestuff/l.

Average specific growth rate:
- from day 0 to day 7, for frond numbers, it was found to be 0.283 in the control and 0.272, 0.263, 0.229, 0.214, 0.196, 0.077 and 0.00 at nominal test concentrations of 0.9, 2.0, 4.2, 9.4, 20.6, 45 and 100 mg dyestuff/l;
- from day 0 to day 7, for biomass (dry weight), it was found to be 0.312 in the control and 0.308, 0.288, 0.263, 0.233, 0.186, 0.143 and 0.00 at nominal test concentrations of 0.9, 2.0, 4.2, 9.4, 20.6, 45 and 100 mg dyestuff/l.

Counting of frond number: on day 7, mean frond numbers per group was 86.8 in control and 80.3, 75.7, 59.7, 53.7, 47.3, 20.7 and 12.0 at nominal test concentrations of 0.9, 2.0, 4.2, 9.4, 20.6, 45 and 100 mg dyestuff/l.

Observation of fronds: all fronds appeared normal on day 0, 3, 6 and on day 7 in control and at nominal tested concentrations of 0.9, 2.0 and 4.2 mg dyestuff/l. Frond appeared normal until day 3 at nominal test concentrations of 9.4, 20.6, 45 and 100 mg dyestuff/l. Some fronds appeared normal and some fronds appeared chlorosis on day 6 and day 7 at the nominal tested concentrations of 9.4, 20.6 and 45 mg dyestuff/l. Fronds appeared chlorosis on day 6 and day 7 at the nominal tested concentration of 100 mg dyestuff/l.

Measurement of dry weight: on day 0 dry weight was found to be 0.0365 g. At the end of the test period, mean dry weight of each group was found to be 0.326 g in control and 0.315, 0.275, 0.231, 0.187, 0.134, 0.100 and 0.027 g at the nominal tested concentration of 0.9, 2.0, 4.2, 9.4, 20.6, 45 and 100 mg dyestuff/l.

DOSE RANGE FINDING
Percent inhibition in growth rate:
- for frond number, it was found to be 0.0, 4.4, 13.2, 24.0, 62.3 and 100 % at nominal tested concentrations of 0.01, 0.1, 1, 10, 50 and 100 mg dyestuff/l;
- for biomass (dry weight), it was found to 0.8, 6.7, 13.9, 23.0, 61.4 and 100.0 % at nominal tested concentrations of 0.01, 0.1, 1, 10, 50 and 100 mg dyestuff/l.

Percent inhibition in yield:
- for frond number, it was found to 0.9, 10.6, 27.4, 44.2, 82.3 and 100 % at the nominal tested concentrations of 0.01, 0.1, 1, 10, 50 and 100 mg dyestuff/l;
- for biomass (dry weight) was found to be 2.0, 16.3, 30.6, 46.4, 84.3 and 100 % were observed at the nominal tested concentrations of 0.01, 0.1, 1, 10, 50 and 100 mg dyestuff/l.

Average specific growth rate:
- from day 0 to day 7, for frond numbers, it was found to be 0.282 in control and 0.282, 0.270, 0.245, 0.215, 0.107 and 0.0 at nominal tested concentrations of 0.01, 0.1, 1, 10, 50 and 100 mg dyestuff/l;
- from day 0 to day 7, for biomass (dry weight), it was found to be 0.340 in the control and 0.338, 0.317, 0.293, 0.262, 0.131 and 0.0 at nominal tested concentrations 0.01, 0.1, 1, 10, 50 and 100 mg dyestuff/l.

Counting of frond number: on day 7, mean frond numbers per group was 65.5 in the control and 65.0, 59.5, 50.0, 40.5, 19.0 and 9.0 at nominal tested concentrations of 0.01, 0.1, 1, 10, 50 and 100 mg dyestuff/l.

Observation of fronds: all fronds appeared normal on day 0, 3, 6 and on day 7 in control and at all nominal test concentrations of 0.01, 0.1, 1 and 10 mg dyestuff/l. Fronds appeared normal until day 3 and some fronds appeared normal and chlorosis on day 6 and 7 at the nominal tested concentration of 50 mg dyestuff/l. Chlorosis was observed on day 3 and chlorosis and desiccation was observed on day 6 and 7 at the nominal tested concentration of 100 mg dyestuff/l.

Measurement of dry weight: on day 0, dry weight was found to be 0.0214 g; at the end, mean dry weight of each group was found to be 0.231 g in control and 0.227, 0.197, 0.167, 0.134, 0.054 and 0.019 g at the nominal tested concentration of 0.01, 0.1, 1, 10, 50 and 100 mg dyestuff/l.

MEASURED CONCENTRATIONS
During the range finding and main study, samples from test concentration were analyzed on day 0 and on day 7. aConcentrations resulted to be within the acceptable range ± 20 % recovery to the nominal concentration.

Results with reference substance (positive control):

The growth inhibition test with Lemna minor exposed to 3,5-Dichlorophenol -97 %-, over 7 days the ErC50 values for growth rate of frond number and biomass (dry weight) were calculated to be 3.66 mg/l and 3.26 mg/l, respectively.
The ErC50 value for the frond number of 3.66 is in good agreement with the results of an international ring test between 2.7 and 3.4 mg/l (n=52).
Hence, the test with toxic standard establishes the acceptability of test system response and test procedures followed.

MAIN TEST

Inhibition in growth rate and yield during the main test

Test conc. (mg dyestuff/l)	Frond number		Dry weight
	% Inhibition in Growth Rate	% Inhibition of in Yield	% Inhibition in Growth Rate	% Inhibition of in Yield
0	-	-	-	-
0.9	3.9	8.7	1.4	3.7
2.0	7.0	14.8	7.7	17.7
4.2	19.0	36.2	15.7	32.9
9.4	24.4	44.2	25.4	48.1
20.6	30.7	52.8	40.5	66.2
45	72.8	88.4	54.1	78.2
100	100	100	100	100

Measured concentrations

Nominal Concentration (mg dyestuff/l)	Recovery (%)
	Day 0	Day 7
0	-	-
0.9	95.26	99.74
2.0	102.26	95.96
4.2	96.98	109.34
9.4	104.98	97.47
20.6	91.41	114.65
45	105.5	92.12
100	94.42	93.41

RANGE-FINDING TEST

Inhibition in growth rate and yield during the range finding study

Test conc. (mg/l)	Frond number		Dry weight
	% Inhibition in Growth Rate	% Inhibition of in Yield	% Inhibition in Growth Rate	% Inhibition of in Yield
0.0	-	-	-	-
0.01	0.0	0.9	0.8	2.0
0.1	4.4	10.6	6.7	16.3
1	13.2	27.4	13.9	30.6
10	24.0	44.2	23.0	46.4
50	62.3	82.3	61.4	84.3
100	100	100	100	100

Measured concentrations

Nominal Concentration (mg dyestuff/l)	Recovery (%)
	Day 0	Day 7
0	-	-
0.01	-	-
0.1	-	-
1	91.19	94.05
10	90.12	82.88
50	93.46	96.26
100	90.59	91.85

Validity criteria fulfilled:

yes

Remarks:

the doubling time of frond number in the control was 2.455 days and 2.442 days, corresponding to average specific growth rate of 0.281 and 0.284 d-1 during range finding study and main study respectively

Conclusions:

ErC50 (7d): 27 mg dyestuff/l (nominal), based on frond number
EyC50 (7d): 10 mg dyestuff/l (nominal), based on frond number

Executive summary:

The test item was assessed for Lemna sp. growth inhibition potential, as per OECD guideline 221.

Lemna minor was exposed to test item by aqueous exposure (treatment) under static condition, over a period of 7 days.

Based on results in a range finding study, the main test was performed at test concentrations of 0.9, 2.0, 4.2, 9.4, 20.6, 45 and 100 mg dyestuff/l, along with control and surrogate groups. Lemna minor were observed for frond number on day 0, 4 and 7. Observation for fronds was made at beginning of test and on day 3, 6 and at end of the test day 7.

During the main study, all fronds appeared normal on day 0, 3, 6 and 7 in control and at nominal test concentrations of 0.9, 2.0 and 4.2 mg dyestuff/l. Fronds appeared normal until day 3 at nominal test concentrations of 9.4, 20.6, 45 and 100 mg dyestuff/l. Some fronds appeared normal and some fronds appeared chlorosis on day 6 and 7 at nominal test concentrations of 9.4, 20.6 and 45 mg/l. Fronds appeared chlorosis on day 6 and day 7 at nominal test concentration of 100 mg dyestuff/l.

ErC50 and EyC50 values for fronds number were found to be 27 and 10 mg dyestuff/l, respectively, with 95 % confidence limits for growth rate of 15.99 and 45.34 mg dyestuff/l and 95 % confidence limits for yield 6.94 and 14.54 mg dyestuff/l, respectively.

ErC50 and EyC50 values for biomass (dry weight) were found to be 32 and 11 mg dyestuff/l, respectively, with 95 % confidence limits for growth rate of 23.23 and 44.01 mg dyestuff/l and 95 % confidence limits for yield 8.86 and 13.20 mg dyestuff/l, respectively.

The NOEC (no observed effect concentration) and LOEC (lowest observed effect concentration) for fronds number were found to be 2.0 and 4.2 mg dyestuff/l, respectively; the NOEC (no observed effect concentration) and LOEC (lowest observed effect concentration) for dry weight (biomass) were found to be 0.9 and 2.0 mg dyestuff/l, respectively.

Conclusion

ErC50 (7d): 27 mg dyestuff/l (nominal), based on frond number

EyC50 (7d): 10 mg dyestuff/l (nominal), based on frond number

Description of key information

ErC50 (7d): 27 mg dyestuff/l (nominal), based on frond number

EyC50 (7d): 10 mg dyestuff/l (nominal), based on frond number

Key value for chemical safety assessment

EC50 for freshwater plants:

27 mg/L

EC10 or NOEC for freshwater plants:

2 mg/L

Additional information

The test item was assessed for lemna sp. growth inhibition potential, as per OECD guideline 221.

Lemna minor was exposed to the test item by aqueous exposure (treatment) under static condition, over a period of 7 days.

Based on results in the range finding study, the main test was performed at test concentrations of 0.9, 2.0, 4.2, 9.4, 20.6, 45 and 100 mg dyestuff/l, along with control and surrogate groups. Lemna minor were observed for frond number on day 0, 4 and 7. Observation for fronds was made at beginning of test and on day 3, 6 and at end of the test day 7.

During the main study, all fronds appeared normal on day 0, 3, 6 and 7 in control and at nominal tested concentrations of 0.9, 2.0 and 4.2 mg dyestuff/l. Fronds appeared normal until day 3 at the nominal tested concentrations of 9.4, 20.6, 45 and 100 mg dyestuff/l. Some fronds appeared normal and some fronds appeared chlorosis on day 6 and 7 at the nominal tested concentrations of 9.4, 20.6 and 45 mg/l. Fronds appeared chlorosis on day 6 and day 7 at the nominal tested concentration of 100 mg dyestuff/l.

The ErC50 and EyC50 values for frond number were found to be 27 and 10 mg dyestuff/l, respectively, with 95 % confidence limits for growth rate of 15.99 and 45.34 mg dyestuff/l and 95 % confidence limits for yield 6.94 and 14.54 mg dyestuff/l, respectively.

The ErC50 and EyC50 values for biomass (dry weight) were found to be 32 and 11 mg dyestuff/l, respectively, with 95 % confidence limits for growth rate of 23.23 and 44.01 mg dyestuff/l and 95 % confidence limits for yield 8.86 and 13.20 mg dyestuff/l, respectively.

The NOEC (No observed effect concentration) and LOEC (Lowest observed effect concentration) for frond number were found to be 2.0 and 4.2 mg dyestuff/l, respectively; the NOEC (No observed effect concentration) and LOEC (Lowest observed effect concentration) for dry weight (Biomass) were found to be 0.9 and 2.0 mg dyestuff/l, respectively.