Reaction products of diazotised 2-amino-5-nitrobenzenesulfonic acid coupled with resorcinol, subsequently coupled with diazotized 2-amino-4-nitro-6-sulfo-phenol, chelated with iron, sodium salt

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 March 28 to April 16, 2018

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:

yes

Details on sampling:

Test item concentrations determined at the beginning of the test, as well as after 3, 5 and 7 days of exposure.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Test item is very soluble, thus test solutions were prepared by respective dilution of a stock solution (test item dissolved in SIS medium) with SIS medium.
Pure SIS medium served as blank control.

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: duckweed.
- Strain: Lemna minor.
- Source: Umweltbundesamt, FGIII 2.5, Überwachungsverfahren Abwasserentsorgung, Schichauweg 58, D-12307 Berlin.
- Method of cultivation: under test conditions.

ACCLIMATION
- Acclimation period: 7-10 days.
- Culturing media and conditions: sterile SIS medium.

Test type:

static

Water media type:

freshwater

Total exposure duration:

7 d

Test temperature:

Target T: 24 ± 2°C - Test T: 20.2 - 23.5°C.

pH:

6.5 ± 0.2 at the beginning of the test; the pH value in the blank control should not increase by more than 1.5 units during the test.

Nominal and measured concentrations:

Range finding test: 1000, 200, 20, 0 mg/L - Definitive test: 200, 100, 50, 25, 12.5, 0 mg/L.

Details on test conditions:

TEST SYSTEM
- Incubation: on a black non-reflecting surface.
- Test vessel: 400 ml beakers, all-glass, with 200 ml of test medium. Beakers were covered with black paper up until the 200 ml mark to ensure that illumination comes only from above and not from the sides.
- No. of fronds per vessel: 9-12 per replicate, 2-4 fronds per plant.
- No. of vessels per concentration: 2 (preliminary test) and 3 (definitive test).
- No. of vessels per control: 3 (preliminary test) and 6 (definitive test).

GROWTH MEDIUM
- Standard medium used: SIS medium.

TEST MEDIUM / WATER PARAMETERS: SIS medium.
Stock solution I (g/L)
NaNO3 = 8.5.
KH2PO4 =1.34.

Stock solution II (g/L)
MgSO4×7H2O = 15.

Stock solution III (g/L)
CaCl2×2H2O = 7.2.

Stock solution IV
Na2CO3 = 4.

Stock solution V
H3BO3 = 1.
ZnSO4×7H2O = 0.05.
Na2MoO4×2H2O = 0.01.
MnCl2×4H2O = 0.2.
Co(NO3)2×6H2O = 0.01.
CuSO4 ×5H2O = 0.005.

Stock solution VI
FeCl3×6H2O = 0.17.
Na2EDTA×2H2O = 0.28.

To prepare 1 l of SIS medium, the following are added to 900 ml of deionised water:
- 10 ml stock solution I
- 5 ml stock solution II, III, IV, VI
- 1 ml stock solution V
The volume is adjusted to 1 l with deionised water. The medium is sterilised before use.

OTHER TEST CONDITIONS
- Adjustment of pH: pH adjusted to 6.5 ± 0.2 with either 0.1 or 1 M HCl or NaOH.
- Illumination:
Intensity: continuous (6500–10000 lux) from Osram Fluora L18W77 (Osram AG, Winterthur, Switzerland) and CH Lighting F18T8/6500K EUP (CH Lighting CO., Ltd., China)
Homogeneity: ± 15 %

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: on day 0, 3, 5, 7.
- Determination of biomass: as dry weight; at day 0 in 6 additional blank replicates and at day 7 in all blanks and test vessels, after drying at 60 °C at least overnight.

RANGE-FINDING STUDY
- Test concentrations: 0, 20, 200, 1000 mg/l.
- Results used to determine the conditions for the definitive study: yes.

Reference substance (positive control):

yes

Remarks:

yearly, with 3,5-dichlorophenol

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

19.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

147 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC10

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: not determined

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Environmental conditions
The pH value in the control drifted by 1.3 units during the whole test period, which is therefore in the range allowed by the guideline (required: not more than 1.5).
The light intensity was 7000-8000 lux (mean 7649 lux; max. variation ± 9 %) at the start of the test and 6990-8020 lux (mean 7675 lux, max. variation ± 9 %) at the end of the exposure period. The light homogeneity was therefore within the required 15 % range.
The temperature ranged between 20.2 and 23.5 °C (i.e. 3.3 °C variation), which is out of the required range of 24 ± 2 °C. Since the growth criterion in the control was fulfilled without a too large variation, this deviation was expected to have no significant impact on the study outcome.
The test item did not show any signs of precipitation, at any of the test concentrations.

Appearance of the plants at the end of the test
In the blank control, the plants were healthy with dark green fronds and the roots reaching to the bottom of the test vessel.
At 12.5 mg/l the plants were mostly dark green with single light green fronds; roots reaching to the bottom of the test vessel.
At 25.0 and 50.0 mg/l the plants were mostly dark green with single light green fronds; the roots were only ½ the length as compared to the roots of the blanks.
At 100 and 200 mg/l most the plants were light green; the roots were only ½ the length as compared to the roots of the blanks.

Results with reference substance (positive control):

According to guideline ISO/CD 20079 “Determination of the toxic effect of water constituents and waste water to duckweed (Lemna minor) — Duckweed growth inhibition test”, the ErC50 value based on the frond number should lie in the range 1.8–3.6 mg/l; this level of sensitivity was attained (data from March 2018).

Test concentrations:

Test concentrations during the 7 -day test period were determined by photometry at the beginning of the test as well as after 3, 5 and 7 days of exposure. These analyses confirmed the right dosage of test item and showed that the concentrations of test item could be satisfactorily maintained. The measured concentrations of test item were 200, 98.0, 48.4, 25.0 and 12.1 mg/l at the beginning of the test and 198, 95.0, 45.4, 22.8 and 10.4 mg/l (i.e. 99, 97, 94, 91 and 86 % of the initial value, respectively) at the end of the test. All concentrations remained within 80-120 % of the nominal value.

Therefore, the effective concentrations (ErC50 and EyC50) were assessed based on the nominal concentration of test item.

 

Effects on frond number:

With respect to growth rate inhibition the following effects as compared to the untreated controls were observed: 22 % at 200 mg/l, 24 % at 100 mg/l, 12 % at 50.0 mg/l and 16 % at 25.0 mg/l. No significant effects were observed at the nominal concentration of 12.5 mg/l.

Based on these effects and the nominal concentrations of test item, the median effect concentration with respect to the growth rate of frond number ErC50 to Lemna minor was estimated to be >200 mg/l. The ErC10 was 19.3 mg/l (95 % confidence limits: 1.96 – 38.7 mg/l); while the NOErC was determined to be 12.5 mg/l.

With respect to yield inhibition the following effects as compared to the untreated controls were observed: 48 % at 200 mg/l, 52 % at 100 mg/l, 31 % at 50.0 mg/l and 39 % at 25.0 mg/l. No significant effects were observed at the nominal concentration of 12.5 mg/l.

Based on these effects and nominal concentrations of test item, the median effect concentration with respect to the yield of frond number EyC50 to Lemna minor was calculated to be 147 mg/l (95% confidence limits: 87.2 – 501 mg/l). The EyC10 was 2.90 mg/l (95% confidence limits: not determined); while the NOErC was determined to be 12.5 mg/l.

 

Effects on dry weight:

With respect to growth rate inhibition the following effects as compared to the untreated controls were observed: 11 % at 200 mg/l, 8 % at 100 mg/l, 6 % at 50.0 mg/l and 13 % at 25.0 mg/l. No significant effects were observed at the nominal concentration of 12.5 mg/l.

Based on these effects and nominal concentrations of test item, the median effect concentration with respect to the growth rate of dry weight ErC50 to Lemna minor was estimated to be >200 mg/l; while the NOErC was determined to be 12.5 mg/l.

With respect to yield inhibition the following effects as compared to the untreated controls were observed: 29 % at 200 mg/l, 24 % at 100 mg/l, 18 % at 50.0 mg/l and 34 % at 25.0 mg/l. No significant effects were observed at the concentration of 12.5 mg/l.

Based on these effects and the nominal concentrations of test item, the median effect concentration with respect to the yield of dry weight EyC50 to Lemna minor was estimated to be >200 mg/l; while the NOEyC was determined to be 12.5 mg/l.

Validity criteria fulfilled:

yes

Conclusions:

ErC50 (7 d) > 200 mg/l for frond number (linear regression with Logit link-function)
ErC50 (7 d) > 200 mg/l for biomass as dry weight (no dose dependent relation found)

Executive summary:

Method:

The growth inhibitory effects of test substance to the duckweed Lemna minor were investigated over a period of 7 days under static conditions, following OECD 221.

The test item is well soluble, i.e. 33.81 g/L. The test solutions were prepared by respective dilutions of a stock solution in SIS medium.

The test was performed at 200, 100, 50, 25, 12.5 mg/L nominal concentrations of test item.

Three parallel test vessels were used for each test concentration of the test item and 6 vessels for the blank controls.

The test concentrations during the 7-day test period were determined by photometry at the beginning of the test, as well as after 3, 5 and 7 days of exposure.

The endpoint frond number was investigated at days 3, 5 and 7 and the endpoint dry weight was determined only at day 7; each of them was assessed as growth rate and yield.

 

Results:

Photometric analyses confirmed the right dosage of the test item, showed that the concentrations were stable over the whole 7-day test period and remained within 80 - 120% of the nominal concentrations. In particular, nominal concentrations of 12.5, 25, 50, 100 and 200 mg/L, corresponding to measured concentrations of 12.1, 25, 48.4, 98 and 200 mg/l at day 0 were 86%, 91%, 94%, 97%, 99% of initial value at day 7.

Therefore, the effective concentrations (E_rC_x and E_yC_x) were assessed based on the nominal concentrations of test item.

EC_x values based on the nominal concentrations of test item were:

parameter expressed as nominal conc. (mg/L) of a.i.		EC50	EC10	NOEC
frond number	growth rate	> 200	19.3	12.5
	yield	147	2.90	12.5
dry weigth	growth rate	>200	n.d	12.5
	yield	>200	n.d.	12.5

The 7 day E_rC₅₀ (based on frond number) of test substance on the duckweed Lemna minor is > 200 mg/L. This value is based on the nominal concentrations of test item. The validity criterion was fulfilled.

Description of key information

E_rC₅₀ (7 d) > 200 mg/l for frond number.

E_rC₅₀ (7 d) > 200 mg/l for biomass as dry weight.

E_yC₅₀ (7 d) = 147 mg/l for frond number.

E_yC₅₀ (7 d) > 200 mg/l for biomass as dry weight.

Additional information

The substance was tested for toxicity on freshwater aquatic plants, i.e. Lemna minor, according to OECD guideline 221 under static exposure conditions.

Concentrations were measured by photometry during the test. These analyses confirmed the right dosage and showed that concentrations were stable over the whole 7-day period and remained within 80 - 120% of the nominal concentrations. Nominal concentrations were 200, 100, 50, 25, 12.5 mg/L of test item.

At the end of the test, appearance of plants showed differences with respect to blank control in a dose dependent way. The tendency was from dark green to light green fronds, with shorter roots.

No signs of precipitation were noted at any concentration.

 

Effects concentrations (EC₁₀ and EC₅₀), based on growth rate and yield were determined for both frond number and dry weight.

No observed effect concentrations (NOEC) were estimated by Williams multiple sequential t-test.

 

The EC₅₀ values after 7 days resulted to be:

- > 200 mg/L, referred to the growth rate of frond number,

- > 200 mg/L, referred to the growth rate of dry weight.