Disodium [3-[4,5-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulphonato(3-)][1-[[2-hydroxy-5-(phenylazo)phenyl]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

Type of information:

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

Adequacy of study:

key study

Study period:

From October 31st to November 14th, 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

No chemical analysis of the test concentrations was conducted. The read across hypothesis has been detailed in the endpoint summary.

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

adopted 23th March 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Source: Umweltbundesamt, D-06813 Dessau-Rosslau, Germany.

ACCLIMATION
- Photoperiod: continuous (6500–7500 lux) from Osram Fluora L18W77 (Osram AG, Winterthur, Switzerland).
- Temperature: 25 ± 1 °C, thermo-controlled room.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

25 ± 1 °C, thermo-controlled room

pH:

The pH-values (measured in the pooled replicates) at the beginning of the test were 5.4 for the blank control, 5.5 at 22.7 mg/l, 5.5 at 227 mg/l and 5.4 at 2270 mg/l. At the end of the test, pH-values were 6.6 for the blank control, 5.7 at 22.7 mg/l, 5.6 at 227 mg/l and 5.5 at 2270 mg/l.

Nominal and measured concentrations:

Nominal: 22.7, 227 and 2270 mg/l

Details on test conditions:

TEST SYSTEM
- Incubation: beakers were covered with black cardboard up the surface of the culture medium and were incubated on a black non-reflecting surface, so that light only comes from above the medium surface. Additionally, the walls of the incubation chambers were covered with black fabric in order to avoid reflection.
- Test vessel: 400 ml beaker, all-glass, with 200 ml of test medium.
- Starting frond number: 9-12 fronds per replicate, 2-4 fronds per plant.
- No. of vessels per concentration: three.
- No. of vessels per control: six.

GROWTH MEDIUM
- Culture: Petri dish containing 300 ml of sterile STEINBERG medium and the exponential growing plant monoculture.

TEST MEDIUM / WATER PARAMETERS
- Test medium: recommended STEINBERG medium.
KNO3: 350 mg/l
KH2PO4: 90 mg/l
K2HPO4 x 3H2O: 16.5 mg/l
MgSO4 x 7H2O: 100 mg/l
Ca(NO3)2 x 4H2O: 295 mg/l
H3BO3: 120.0 µg/l
ZnSO4 x 7H2O: 180.0 µg/l
Na2MoO4 x 2H2O: 44.0 µg/l
MnCl2 x 4H2O: 180.0 µg/l
FeCl3 x 6H2O: 760.0 µg/l
C10H14N2Na2O8 x 2H2O: 1500.0 µg/l

OTHER TEST CONDITIONS
- Adjustment of pH: the pH of the STEINBERG medium was adjusted before the test (pH 5.5 ± 0.2).
- Photoperiod: continuous (6500–7500 lux) from Osram Fluora L18W77 (Osram AG, Winterthur, Switzerland).

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

22.7 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

227 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

EFFECTES on FROND NUMBERS
With respect to growth rate inhibition for the endpoint frond numbers, the following effects as compared to the untreated controls were observed: 91 % at 2270 mg/l, 65 % at 227 mg/l and 15 % at 22.7 mg/l.
With respect to yield inhibition for the endpoint frond numbers, the following effects as compared to the untreated controls were observed: 97 % at 2270 mg/l, 85 % at 227 mg/l and 30 % at 22.7 mg/l.
Based on these data the median effect concentrations of the test item to Lemna minor with respect to growth rate (ErC50) and yield (EyC50) for the endpoint frond numbers were estimated to be 22.7–227 mg/l nominal concentration.
The no-observed-effect concentrations to Lemna minor with respect to growth rate (NOErC) and yield (NOEyC) for the endpoint frond numbers were <22.7 mg/l nominal concentration, as determined by Dunnett's test.

EFFECTS on DRY WEIGHT
With respect to growth rate inhibition for the endpoint dry weight, the following effects as compared to the untreated controls were observed: 100 % at 2270 mg/l, 64 % at 227 mg/l and 19 % at 22.7 mg/l.
With respect to yield inhibition for the endpoint dry weight, the following effects as compared to the untreated controls were observed: 100 % at 2270 mg/l, 84 % at 227 mg/l and 38 % at 22.7 mg/l.
Based on these data the median effect concentrations of the test item to Lemna minor with respect to growth rate (ErC50) and yield (EyC50) for the endpoint dry weight were estimated to be 22.7–227 mg/l nominal concentration.
The no-observed-effect concentrations to Lemna minor with respect to growth rate (NOErC) and yield (NOEyC) for the endpoint dry weight were <22.7 mg/l nominal concentration, as determined by Dunnett's test.

In the control and at 22.7 mg/l the plants looked healthy. At 227 mg/l the plants were smaller and partially presented signs of chlorosis, the roots were much shorter; older plants presented signs of necrosis. At 2270 mg/l the plants were very small, almost entirely necrosed and almost without roots.

Frond number between days 0 and 7 and the corresponding growth rate inhibition

Nominal Concentration	Replicate	Frond Number, Data				Frond Number, Growth Rate
		d 0	d 3	d 5	d 7	GRa^a)μ7-0 (d^-1)	Doubling time (days)^b)	Inhibition (%)	Inhibition mean value (%)
(mg/l)
Control	A	10	24	46	85	0.31	2.27	-2.4	0.0
	B	10	24	43	90	0.31	2.21	-5.1
	C	10	23	39	74	0.29	2.42	4.2
	D	10	21	41	71	0.28	2.48	6.2
	E	10	26	47	79	0.30	2.35	1.1
	F	10	24	56	88	0.31	2.23	-4.1
22.7	A	10	20	36	51	0.23	2.98	22.0	14.9
	B	10	22	44	68	0.27	2.53	8.3
	C	10	23	35	60	0.26	2.71	14.3
227	A	10	13	19	22	0.11	6.15	62.3	64.5
	B	10	16	20	21	0.11	6.54	64.5
	C	10	13	18	20	0.10	7.00	66.8
2270	A	10	11	12	12	0.03	26.61	91.3	91.4
	B	10	10	11	11	0.01	50.91	95.4
	C	10	10	12	13	0.04	18.49	87.4

a) average specific growth rate over 7 days

b) average doubling time of controls must be < 2.5 d. Here, doubling time was 2.3 days

Frond number between days 0 and 7 and the corresponding yield inhibition

Nominal Concentration	Replicate	Frond Number, Data				Frond Number, Yield
		d 0	d 3	d 5	d 7	Yield days 0-7	Inhibition (%)	Inhibition mean value (%)
(mg/l)
Control	A	10	24	46	85	75	-5.4	0.0
	B	10	24	43	90	80	-12.4
	C	10	23	39	74	64	10.1
	D	10	21	41	71	61	14.3
	E	10	26	47	79	69	3.0
	F	10	24	56	88	78	-9.6
22.7	A	10	20	36	51	41	42.4	30.2
	B	10	22	44	68	58	18.5
	C	10	23	35	60	50	29.7
227	A	10	13	19	22	12	83.1	84.5
	B	10	16	20	21	11	84.5
	C	10	13	18	20	10	85.9
2270	A	10	11	12	12	2	97.2	97.2
	B	10	10	11	11	1	98.6
	C	10	10	12	13	3	95.8

Dry Weight at days 0 and 7 and the corresponding growth rate inhibition

Nominal Concentration	Replicate	Dry Weight, Data		Dry Weight, Growth Rate
		d 0^a)(mg)	d 7 (mg)	GRa^b)μ7-0 (d^-1)	Inhibition (%)	Inhibition mean value (%)
(mg/l)
Control	A	1.4	11.9	0.31	0.5	0.0
	B	1.4	12.9	0.32	-3.3
	C	1.4	10.4	0.29	6.7
	D	1.4	11.1	0.30	3.7
	E	1.4	12.5	0.31	-1.8
	F	1.4	13.6	0.32	-5.8
22.7	A	1.4	6.4	0.22	29.3	19.3
	B	1.4	9.2	0.27	12.4
	C	1.4	8.5	0.26	16.1
227	A	1.4	3.1	0.11	63.0	63.5
	B	1.4	3.1	0.11	63.0
	C	1.4	3	0.11	64.6
2270	A	1.4	1.3	-0.01	103.4	100.1
	B	1.4	1.5	0.01	96.8
	C	1.4	1.4	0.00	100.0

a) average of six frond samples similar to those used in the test

b) average specific growth rate over 7 days.

Dry Weight at days 0 and 7 and the corresponding yield inhibition

Nominal Concentration	Replicate	Dry Weight, Data		Dry Weight, Yield
		d 0^a)(mg)	d 7 (mg)	Yield days 0-7	Inhibition (%)	Inhibition mean value (%)
(mg/l)
Control	A	1.4	11.9	10.50	1.6	0.0
	B	1.4	12.9	11.50	-7.8
	C	1.4	10.4	9.00	15.6
	D	1.4	11.1	9.70	9.1
	E	1.4	12.5	11.10	-4.1
	F	1.4	13.6	12.20	-14.4
22.7	A	1.4	6.4	5.00	53.1	37.8
	B	1.4	9.2	7.80	26.9
	C	1.4	8.5	7.10	33.4
227	A	1.4	3.1	1.70	84.1	84.4
	B	1.4	3.1	1.70	84.1
	C	1.4	3.0	1.60	85.0
2270	A	1.4	1.3	-0.10	100.9	100.0
	B	1.4	1.5	0.10	99.1
	C	1.4	1.4	0.00	100.0

a) average of six frond samples similar to those used in the test

Validity criteria fulfilled:

yes

Remarks:

the doubling time of frond number in the control was be less than 2.5 days (60 h), corresponding to approximately a 7-fold increase in 7 days and an average specific growth rate of 0.275/d

Conclusions:

ErC50 (7d): 22.7 mg/l (nominal)
EyC50 (7d): 227 mg/l (nominal)

Executive summary:

Method

The inhibitory effects based on the average specific growth rate and the yield (absolute increase in plant size) of the test item to the duckweed Lemna minor was investigated over a period of 7 days. The test was performed at concentrations of 20, 200 and 2000 mg/l of the active ingredient. Taking into account a purity of 88.02 %, nominal concentrations of 22.7, 227 and 2270 mg/l, respectively, were prepared.

No chemical analysis of the test concentrations was conducted. The determination of the effect concentrations was based on the nominal concentrations, assuming the test item to be stable in water for the whole test period.

Results

With respect to growth rate inhibition for the endpoint frond numbers, the following effects as compared to the untreated controls were observed: 91 % at 2270 mg/l, 65 % at 227 mg/l and 15 % at 22.7 mg/l.

With respect to yield inhibition for the endpoint frond numbers, the following effects as compared to the untreated controls were observed: 97 % at 2270 mg/l, 85 % at 227 mg/l and 30 % at 22.7 mg/l.

Based on these data the median effect concentrations of the test item to Lemna minor with respect to growth rate (ErC50) and yield (EyC50) for the endpoint frond numbers were estimated to be 22.7–227 mg/l nominal concentration.

The no-observed-effect concentrations to Lemna minor with respect to growth rate (NOErC) and yield (NOEyC) for the endpoint frond numbers were < 22.7 mg/l nominal concentration, as determined by Dunnett's test.

With respect to growth rate inhibition for the endpoint dry weight, the following effects as compared to the untreated controls were observed: 100 % at 2270 mg/l, 64 % at 227 mg/l and 19 % at 22.7 mg/l.

With respect to yield inhibition for the endpoint dry weight, the following effects as compared to the untreated controls were observed: 100 % at 2270 mg/l, 84 % at 227 mg/l and 38 % at 22.7 mg/l.

Based on these data the median effect concentrations of the test item to Lemna minor with respect to growth rate (ErC50) and yield (EyC50) for the endpoint dry weight were estimated to be 22.7–227 mg/l nominal concentration.

The no-observed-effect concentrations of the test item to Lemna minor with respect to growth rate (NOErC) and yield (NOEyC) for the endpoint dry weight as were < 22.7 mg/l nominal concentration, as determined by Dunnett's test.

Description of key information

ErC50 (7d): 22.7 mg/l (nominal)
EyC50 (7d): 227 mg/l (nominal)

Key value for chemical safety assessment

EC50 for freshwater plants:

22.7 mg/L

Additional information

There are no available data on toxicity to invertebrates of Acid Brown 298, thus a read across approach has been followed.

The Read across approach here discussed is based on the structural similarity of the two main components of target and source substances, on their similar composition and biodegradation potential, in order to fulfil eco-toxicological information requirements (toxicity to aquatic plants other than algae). Details in attachment.

The inhibitory effects based on the average specific growth rate and the yield (absolute increase in plant size) of the Similar Substance 01 to the duckweed Lemna minor was investigated over a period of 7 days. The test was performed at concentrations of 20, 200 and 2000 mg/l of the active ingredient. Taking into account a purity of 88.02 %, nominal concentrations of 22.7, 227 and 2270 mg/l, respectively, were prepared.

No chemical analysis of the test concentrations was conducted. The determination of the effect concentrations was based on the nominal concentrations, assuming the test item to be stable in water for the whole test period.

With respect to growth rate inhibition for the endpoint frond numbers, the following effects as compared to the untreated controls were observed:

91 % at 2270 mg/l, 65 % at 227 mg/l and 15 % at 22.7 mg/l.

With respect to yield inhibition for the endpoint frond numbers, the following effects as compared to the untreated controls were observed:

97 % at 2270 mg/l, 85 % at 227 mg/l and 30 % at 22.7 mg/l.

Based on these data the median effect concentrations of the test item to Lemna minor with respect to growth rate (ErC50) and yield (EyC50) for the endpoint frond numbers were estimated to be 22.7–227 mg/l nominal concentration.

ErC50 = 22.7 mg/l; EyC50 = 227 mg/l

The no-observed-effect concentrations to Lemna minor with respect to growth rate (NOErC) and yield (NOEyC) for the endpoint frond numbers were < 22.7 mg/l nominal concentration, as determined by Dunnett's test.

With respect to growth rate inhibition for the endpoint dry weight, the following effects as compared to the untreated controls were observed:

100 % at 2270 mg/l, 64 % at 227 mg/l and 19 % at 22.7 mg/l.

With respect to yield inhibition for the endpoint dry weight, the following effects as compared to the untreated controls were observed:

100 % at 2270 mg/l, 84 % at 227 mg/l and 38 % at 22.7 mg/l.

Based on these data the median effect concentrations of the test item to Lemna minor with respect to growth rate (ErC50) and yield (EyC50) for the endpoint dry weight were estimated to be 22.7–227 mg/l nominal concentration.

The no-observed-effect concentrations of the test item to Lemna minor with respect to growth rate (NOErC) and yield (NOEyC) for the endpoint dry weight as were < 22.7 mg/l nominal concentration, as determined by Dunnett's test.

Results:

ErC50 (7d): 22.7 mg/l (nominal)
EyC50 (7d): 227 mg/l (nominal)