Reaction mass of sodium 4-(4-acetamidophenylamino)-1-amino-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate and hydrogen 4-(4-acetamidophenylamino)-1-amino-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

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 ferbruary 10 to 17, 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

Deviations:

yes

Remarks:

The temperature ranged between 18.4 and 26.4°C (i.e. 6.0°C variation), which is out of the required range of 24 ± 2 °C.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 611, 193, 61.1, 19.3 and 6.11 mg/l
- Sampling method: test solutions prepared by respective dilutions of a stock solution (test item dissolved in SIS Medium with SIS Medium)
- Sample storage conditions before analysis: at room temperature, protected from light

Vehicle:

no

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: Lemna minor
- Source (laboratory, culture collection): Umweltbundesamt, FGIII 2.5, Uberwachungsverfahren Abwasserentsorgung, Schichauweg 58, D-12307 Berlin
- Age of inoculum (at test initiation):
- Method of cultivation:

ACCLIMATION
- Acclimation period: 7 - 10 days under the conditions of the test
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed:

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

Ranged between 18.4 and 26.4°C

pH:

6.5 at the beginning of the test

Nominal and measured concentrations:

611, 193, 61.1, 19.3 and 6.11 mg/l nominal concentration (corresponding to 500, 158.1, 50.0, 15.8 and 5.00 mg/l of the active ingredient)

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: beakers were incubated on a black non-reflecting surface
- Test vessel: beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 400 ml beakers, all-glass, with 200 ml of test medium
- Type of cover: black paper
- No. of fronds per vessel: 9-12
- No. of fronds per colony: 2-4
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: pH is adjusted to 6.5 ± 0.2 with either 0.1 or 1 M HCl or NaOH
- Photoperiod:
- Light intensity and quality: 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: not specified
- Determination of biomass: dry weight
- Determination of frond area: no


RANGE-FINDING STUDY
- Test concentrations: 10, 100 and 500 mg/l
- Results used to determine the conditions for the definitive study: at 10 mg/l 3.4 % inhibition based on growth rate, at 100 mg/l 35 % inhibition, at 500 mg/l 59 % inhibition.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 615 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: based on frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 197 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Remarks on result:

other: based on frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 889 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: based on Dry weight

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 178 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Remarks on result:

other: based on Dry weight

Test concentrations

The test concentrations during the 7-day test period weredetermined byphotometryat the beginning of the test, as well as after 3, 5 and 7 days of exposure.These analyses confirmed the right dosage of the test item, and showed that the concentrations of the test item decreased over the whole 7-day test period. The measured concentrations of the active ingredient at the beginning of the test were 514, 163, 49.3, 16.0 and 4.93 mg/l and 345, 113, 50.6, 14.3 and 4.24 mg/l (i.e. 67, 69, 103, 90 and 86 %, respectively, of the initial value) after 7 days.

Therefore, the effective concentrations (E_rC₅₀and E_yC₅₀) were assessed based on the geometric mean (GM) of the measured concentrations of the active ingredient.

Effects on frond number

With respect to growth rate inhibition, the following effects as compared to the untreated controls were observed: 39 % at 500 mg/l and 23 % at 158 mg/l. No significant effects were observed at the concentrations 50.0, 15.8 and 5.00 mg/l.

Based on these effects and the measured concentrations of the active ingredient, the median effect concentration with respect to the frond number’s growth rate (frond number E_rC₅₀) of the test substance to Lemna minor was calculated to be 615 mg/l (95 % confidence limits: 439–1115 mg/l). The E_rC₁₀was 62.0 mg/l (29.6–92.3 mg/l); while the NOE_rC was determined to be 49.1 mg/l.

With respect to yield inhibition, the following effects as compared to the untreated controls were observed: 66 % at 500 mg/l and 46 % at 158 mg/l. No significant effects were observed at the concentrations 50.0, 15.8 and 5.00 mg/l.

Based on these effects and the measured concentrations of the active ingredient, the median effect concentration with respect to the frond number’s yield (frond number E_yC₅₀) of the test substance to Lemna minor was calculated to be 197 mg/l (95 % confidence limits: 141–300 mg/l). The E_yC₁₀was 29.2 mg/l (9.26–50.6 mg/l); while the NOE_yC was determined to be 49.1 mg/l.

Effects on dry weight

With respect to growth rate inhibition, the following effects as compared to the untreated controls were observed: 34 % at 500 mg/l and 23 % at 158 mg/l. No significant effects were observed at the concentrations 50.0, 15.8 and 5.00 mg/l.

Based on these effects and the measured concentrations of the active ingredient, the median effect concentration with respect to the dry weight’s growth rate (dry weight E_rC₅₀) of the test substance to Lemna minor was calculated to be 889 mg/l (95 % confidence limits: 546–2429 mg/l). The E_rC₁₀was 57.5 mg/l (22.5–91.2 mg/l); while the NOE_rC was determined to be 49.1 mg/l.

With respect to yield inhibition, the following effects as compared to the untreated controls were observed: 65 % at 500 mg/l and 52 % at 158 mg/l. No significant effects were observed at the concentrations 50.0, 15.8 and 5.00 mg/l.

Based on these effects and the measured concentrations of the active ingredient, the median effect concentration with respect to the dry weight’s yield (dry weight E_yC₅₀) of the test substance to Lemna minor was calculated to be 178 mg/l (95 % confidence limits: 124–280 mg/l). The E_yC₁₀was 20.8 mg/l (6.01–38.0 mg/l); while the NOE_yC was determined to be 49.1 mg/l.

Environmental conditions

The pH value in the control drifted by 0.6 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 7500-8250 lux (mean 7939 lux; max. variation ± 6 %) at the start of the test and 6950-8340 lux (mean 7663 lux, max. variation ± 9 %) at the end of the exposure period. The light homogeneity was therefore in the required ± 15 % range.

The temperature ranged between 18.4 and 26.4 °C (i.e. 6.0 °C variation), which is out of the required range of 24 ± 2 °C. Since the test vessels were randomly re-placed, since all test vessels were exposed to the same temperature, since the toxicity is calculated based on the blank control, and since the growth criterion in the control was fulfilled without a too large variation, this deviation is not expected to have a significant impact on the outcome of the study.

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 roots reaching to the bottom of the test vessel.

At 5.00 mg/l, the plants looked like the blanks, with dark green fronds and roots reaching to the bottom of the test vessel.

At 15.8 and 50.0 mg/l, the plants were healthy with dark green fronds and roots reaching to the bottom of the test vessel; the plants were slightly smaller compared to the blank control.

At 158 mg/l, the fronds were dark green and somehow misshapen; the size of the roots were only two thirds of the blank control and the plants were significantly smaller as compared to the blank controls.

At 500 mg/l, the plants werevery small, with plants and fronds partly blue discolored; the size of the roots were only two thirds of the blank control.

Validity criteria fulfilled:

yes

Conclusions:

ErC50 = 615 mg/l based on frond number
EyC50 = 197 mg/l based on frond number
ErC50 = 889 mg/l based on dry weight
EyC50 = 178 mg/l based on dry weight

Executive summary:

Method

The inhibitory effects ofthe test substance the duckweed Lemna minor were investigated over a period of 7 days, based on the frond number and biomass (dry weight), following the guideline OECD 221.

The test solutions were prepared by respective dilutions of a stock solution in Swedish standard-Medium (SIS Medium).

The test was performed at 611, 193, 61.1, 19.3 and 6.11 mg/l nominal concentration of the test item, corresponding to 500, 158.1, 50.0, 15.8 and 5.00 mg/l of the active ingredient.

Three parallel test vessels were used for each test concentration of the test item and six 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. These analyses confirmed the right dosage of the test item, and showed that the concentrations of the loadings decreased over the whole 7-day test period after 7 days and did not remain within 80-120% range of the initial value (67, 69, 103, 90 and 86%, respectively, of the initial value). Therefore, the effective concentrations (E_rC₅₀and E_yC₅₀) were assessed based on the geometric mean (GM) of the measured concentrations of the active ingredient.

The two endpoints frond number and biomass (dry weight) were investigated at days 3, 5 and 7, and each of them were assessed as growth rate and yield.

Results

ErC50 = 615 mg/l based on frond number

EyC50 = 197 mg/l based on frond number

ErC50 = 889 mg/l based on dry weight

EyC50 = 178 mg/l based on dry weight

Description of key information

ErC50 = 615 mg/l based on frond number