Hydrogen [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(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 analogue substance

Adequacy of study:

key study

Study period:

21 Mar 2016 to 14 April 2016

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)

Version / remarks:

2006

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Remarks:

No analytical method is available for the multi-constituent test substance. See 'Details on analytical methods' for further information.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Preliminary testing: A preliminary WSF study was conducted (non-GLP) to determine the appropriate WSF stirring time, the efficacy of filtration, and to observe any changes in the test solutions during static test conditions. Results of this study indicated that 48 hour WSF stirring, filtration of test solutions, and daily water renewals were necessary.
- Method: Water soluble fractions (WSFs) were prepared at 6.25, 12.5, 25, 50, and 100 mg/L nominal loading rates and tested alongside a laboratory (negative) control and a WSF (method) control. A total of seven sets of WSFs were prepared every ~24 hours as needed to allow for daily water renewals. To prepare the WSFs, the Test Item was weighed and added directly to 1.5L of Lemna (SIS) test media in a 2L glass vessel. Two litres of the WSF (method) control (0 mg/L) were prepared alongside the Test Item WSFs; however, no test item was added. Each solution was stirred on a stirplate using a magnetic stir bar to create a vortex (~10%) and covered by Plexiglas. Stirring occurred for approximately 47 hours, and solutions were allowed to settle for approximately 1 hour. WSF solutions were removed from the mixing vessel and collected in a glass holding vessel (e.g., 2 L glass beaker) by siphoning from the middle of the water column with a Teflon tube, discarding the first ~50 – 100 mL, into an appropriate container. Each siphoned WSF solution was filtered into a glass media bottle fitted with a sterile bottle top 0.2 μm filter. The WSF (method) control was filtered first, followed by each Test Item loading rate up to 25 mg/L. An additional filter unit was used for the 50 mg/L and 100 mg/L test loading rates. The filters were pre-saturated with ~100 mL of each test solution, and the resulting filtrate discarded, prior to collecting each test solution in the appropriate media bottle.
- Application: Each test vessel was rinsed with the appropriate test solution before the ~125 mL test volume was dispensed. Three replicate test vessels of each test item loading rate were prepared. Six replicate test vessels were prepared for the WSF (method) control and for the laboratory (negative) control.
- Evidence of undissolved material: At the time of preparation and through to 48 hours, the WSF control appeared clear and colourless. The Test Item WSFs increased in colour, intensity, and opacity as Test Item loading rate increased. The lower loading rate WSFs appeared light grey/purple and translucent, with black particles visible on the water surface and in the water column. The higher loading rates appeared very dark grey/black and opaque, with fine black particles visible on the water surface. In each of the WSFs containing Test Item, the Test Item appeared to creep up the sides of the glass beaker at time of addition. After ~24 hours, the 12.5 mg/L to 100 mg/L WSFs appeared to have a shiny oil-like swirl on the water surface, concentrated at the vortex. After filtration, the Test Item WSF solutions appeared significantly lighter and were all translucent, with no visible particles or oil-like swirl.

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: Lemna minor
- Source: Canadian Phycological Culture Centre, Waterloo, ON, Canada.
- Age of organism at test initiation: 8 days old
- Method of cultivation: The test plants were cultured in lab for >3 weeks before use under the same conditions as used for the test. Plants were from an axenic culture, maintained on a weekly basis by transferring to sterile growth medium. The test culture was transferred to sterile growth medium 8 days prior to test initiation.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

24 - 26 °C

pH:

6.8 - 7.5

Nominal and measured concentrations:

- Nominal loading rates: 0 (control), 6.25, 12.5, 25, 50, and 100 mg test item/L

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: Yes
- Test vessel: 250 mL Erlenmeyer flask; sealed with foam plugs
- Fill volume: ~125 mL
- Aeration: No
- Agitation: No
- Renewal rate of test solution: Daily
- No. of fronds per replicate: 9
- Mean initial frond weight: 0.74 mg/replicate
- No. of vessels per concentration (replicates): 3 replicates for Test Item concentrations (loading rates)
- No. of vessels per control (replicates): 6 replicates for each control,
- Controls: Laboratory (negative) control (dilution water only) and WSF (method) control
- Randomisation: Placing on shelf in the test room using a randomisation pattern

GROWTH MEDIUM
- Standard medium used: Yes, Synthetic growth medium; Swedish Standard (SIS)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic growth medium; Swedish Standard (SIS)
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature daily in monitoring vessel; pH at beginning and end of each renewal period; Total organic carbon (TOC) was measured (not according to GLP) as an additional water quality parameter on initial and final test solutions prepared for test initiation and for the final water renewal.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: The pH of the SIS media was 8.9 and was adjusted to 6.4 using 6N HCl prior to sterilization and use. No correction of pH during preparation of test solutions.
- Photoperiod: Continuous 24-h light
- Light intensity and quality: 99 - 118 μmol/m²/s

EFFECT PARAMETERS MEASURED:
- Determination of frond number: Manual counting on day 3, 5 and at test completion (day 7)
- Determination of biomass: Dry weight (dried for a minimum of 24 hours at ~60 °C) at test initiation (representative sample) and completion (day 7)
- Other: Observations on Lemna and test solution appearance were also recorded on the same days as frond counts.

RANGE-FINDING STUDY
- Test concentrations: Nominal loading rate range of 0, 0.1, 1.0, 10, and 100 mg Test Item/L
- Results used to determine the conditions for the definitive study: ELR50 for frond increase based on nominal loading rates for the preliminary test was >100 mg Test Item/L

Reference substance (positive control):

yes

Remarks:

nickel sulphate hexahydrate

Key result

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOELR

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

other: yield

Duration:

7 d

Dose descriptor:

NOELR

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

other: yield

Details on results:

FROND NUMBER AND DRY WEIGHT
A decrease in frond numbers and dry weight was visible with increasing test item loading rate, where significant inhibition was observed from a loading rate of 50 mg/L. For details see 'Any other information on results incl. tables'.

OBSERVATIONS ON TEST SOLUTIONS
At test initiation, the laboratory (negative) control test solutions to the 25 mg/L loading rate solutions appeared clear and colourless. The 50 mg/L loading rate test solution appeared very light purple and translucent. The 100 mg/L loading rate test solution appeared to be a slightly darker grey/purple and translucent. The test solution colour remained unchanged after approximately 24 hours, when compared to the new solutions used for the water renewal. Particulates were not visible in the initial or final test solutions over the course of the test.

OBSERVATIONS ON PLANTS
At test completion, plants in the laboratory control up to the 25 mg/L test loading rate appeared light green, healthy, with some transparent fronds. Plants in the 50 mg/L loading rate appeared light green and healthy with darkened roots and some transparent fronds. Plants in the 100 mg/L loading rate appeared smaller, dark green, and healthy with darkened roots and some transparent fronds.

OTHER FINDINGS
- Water quality: The initial pH measurements did not differ from the final measurements by more than 1.5 units. TOC concentrations increased in a dose related manner with loading rate. For lab control (0), WSF control (0), 6.25, 12.5, 25, 50 and 100 mg/L loading rate the average TOC concentration measured was 1.14, 0.92, 0.98, 1.23, 2.16, 3.53 and 8.59 mg C/L, respectively.

Results with reference substance (positive control):

The 7-day EC25 was 6.90 mg Ni/L (95% confidence limits: 4.09 - 10.94 mg Ni/L) for the reference item. This value was within the acceptable range (± 2 SD) of previous tests conducted in this laboratory and indicates that the test organisms responded normally to the reference item.

Reported statistics and error estimates:

The 7-day ELR50 with 95% confidence limits for yield and average specific growth rate, based upon Lemna frond number and dry weight were calculated using a computerised statistical package, CETIS™ (Version 1.8.7.16), a Comprehensive Environmental Toxicity Information System, recommended by Environment Canada. NOELR and LOELR for the 7-day test were also calculated. The test endpoints were calculated and reported based on the nominal test item loading rate.

 Table: Mean yield and specific growth rate based on frond number and dry weight

Nominal Loading Rate (mg/L)	Frond Number (Day 0 - 7)						Dry Weight (Day 0 - 7)
	Mean Yield	SD	% CV	Mean SGR	SD	% CV	Mean Yield (mg)	SD (mg)	% CV	Mean SGR	SD	% CV
Lab Control (0)	123.5	9.4	7.6	0.38	0.01	2.6	11.07	1.18	10.7	0.39	0.01	3.4
WSF Control (0)	135.3	8	5.9	0.4	0.01	2	11.46	0.90	7.9	0.40	0.01	2.7
625	137.7	7	5.1	0.4	0.01	1.7	11.69	0.71	6.1	0.40	0.01	2.1
125	128.7	11	8.6	0.39	0.01	3	11.84	0.67	5.7	0.40	0.01	1.9
25	122.7	3.5	2.9	0.38	0	1	11.24	0.41	3.6	0.40	0.00	1.2
50	96.7	11.2	11.5	0.35	0.02	4.4	9.19	1.09	11.9	0.37	0.02	4.4
100	76.7	8.7	11.4	0.32	0.01	4.6	7.55	0.81	10.7	0.34	0.01	4.2

SGR: Specific Growth Rate

SD: Standard Deviation

% CV: Percent Coefficient of Variation

Table: Inhibition of yield and growth rate of Lemna after 7 days

Nominal loading rate [mg/L]	Mean % inhibition of yield*		Mean % inhibition of growth rate*
	Based on frond number	Based on dry weight	Based on frond number	Based on dry weight
Lab Control (0)	N/A	N/A	N/A	N/A
Lab Control (0)	-9.6	-3.6	-3.2	-1.2
6.25	-11.5	-5.6	-3.8	-2.0
12.5	-4.2	-7.0	-1.4	-2.4
25	0.7	-1.6	0.2	-0.6
50	21.7	16.9	8.5	6.3
100	37.9	31.8	16.3	12.8

N/A: Not Applicable

*A negative % inhibition value represents stimulation, when compared to the lab (negative) control

Table: Obtained effect concentrations after 7 days of exposure – Yield

	Nominal test item loading rate (mg/L)
	ELR50 (95% CL)	NOELR	LOELR
Frond number	>100 (N/A)	25	50
Dry weight	>100 (N/A)	25	50

 

Table: Obtained effect concentrations after 7 days of exposure – Specific growth rate

	Nominal test item loading rate (mg/L)
	ELR50 (95% CL)	NOELR	LOELR
Frond number	>100 (N/A)	25	50
Dry weight	>100 (N/A)	25	50

Validity criteria fulfilled:

yes

Conclusions:

Not toxic to aquatic plants (Lemna Minor)
EC50 > 100 mg/l

Description of key information

In duckweed (L. gibba), the 7-d ErL50 (growth rate, based on frond number) and 7-d EyL50 (yield, based on frond number) are >100 mg/L and >100 mg/L, respectively on the analogue substance. The 7-d NOELR values for growth rate and yield are 25 mg/L. These effect values are based on nominal test item loading rates.

Based on the read across considerations same results apply to the target substance.

Additional information

The toxicity to aquatic plants other than algae was determined in a study according to OECD TG 221 and in compliance with GLP criteria (Stahl, 2016) performed on the analogue substance. In this study, 9 fronds of uniform, healthy-looking duckweed (L. gibba) were introduced in test vessels and exposed to the following range of nominal loading rates: 6.25, 12.5, 25, 50, and 100 mg test item /L, along with a water soluble fraction (WSF) method control (dilution water only, 0 mg test item/L) and a laboratory (negative) control (dilution water only). The test was performed in 3 replicates per test concentration (6 replicates for each control). Plants were exposed for 7 days under semi-static conditions (test medium renewal every 24 hours). Test concentrations were not analytically verified because a reliable method for analysis was not available. Since all reasonable efforts were taken to produce a WSF of the test item in the test media, results are based on the loading rate. Observations of frond number and appearance (of Lemna and test solution) were made on determination day 3 and 5 and at the end of the exposure. At the end of the test period, dry weight was determined. A decrease in frond numbers and dry weight was visible with increasing test item loading rate, where significant inhibition was observed from a loading rate of 50 mg/L. Darkening of roots was observed from a test item loading rate of 50 mg/L; effects on plant development were visible at the highest test item loading rate (100 mg/L). Appearance of the test solutions remained unchanged over 24 hours and ranged from clear and colourless (up to 25 mg/L loading rate) to very light purple and translucent (50 mg/L loading rate) to slightly darker grey/purple and translucent (100 mg/L loading rate). The ECL50-values for inhibition of the specific growth rate of fronds (ErL50) and dry weight (ErdwL50) were >100 mg/L and >100 mg/L, respectively. The EL50-values for inhibition of yield (EyL50) and dry weight inhibition of yield (EydwL50) were >100 mg/L and >100 mg/L, respectively. The NOELR values for growth rate and yield, based on frond numbers, are 25 mg/L.