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Toxicity to aquatic plants other than algae

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Reference
Endpoint:
toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 28th August 2018 to 14th September 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:
March 2006
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EC 761/2009
Version / remarks:
Commission Regulation (EU) 2016/266 of 7 December 2015 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council. C.26. Lemna sp. Growth Inhibition Test (O.J. L 54 of 1.3.2016).
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
TEST ITEM
- identification: Acid Brown 396:1
- Lot/batch No.: Lot no. 77171
- ACH sample no. M1807-07566
- Purity: 100%
- Physical form: solid

STABILITY AND STORAGE CONDITIONS OF TEST ITEM
- Storage condition: at room temperature, protected from light
- Solubility: the test item is soluble in the test water.
Analytical monitoring:
yes
Remarks:
pH
Details on sampling:
CONCENTRATIONS
The test concentrations of Acid Brown 396:1 during the 7-days test period were determined by photometry at the beginning of the test as well as after 2, 5 and 7 days of exposure. These analyses confirmed the right dosage of the test item and showed that the concentrations were satisfactorily maintained. The measured concentrations remained within 80–120% of the nominal value, respectively 500, 158, 50.0, 15.8 and 5.00 mg/l.
Therefore, the effective concentrations ECx were assessed based on the nominal concentrations of the test item.

SAMPLING
The following test vessels were set up:
- Test solution (Tn); containing test water, test item and duckweed (three replicates per concentration);
- Blank control (Bn); containing test water and duckweed (six replicates).
Since the test item is soluble, the test solutions were prepared by respective dilutions of a stock solution (test item dissolved in test water) with test water. Young and rapidly growing plants without visible lesions or discoloration (chlorosis) from an exponentially growing monoculture of Lemna minor were distributed into the test vessels. These vessels were randomly placed in the incubation chamber at the start of the test and randomly repositioned after the observations made at days 2 and 5.

SAMPLE STORAGE CONDITIONS BEFORE ANALYSIS
Samples were taken from all test solutions including the control at the beginning of the test and after 2, 5 and 7 days of exposure. For sampling at day 2, 5 and 7, the replicates per treatment were pooled prior to sampling. All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis. In pre-experiments for investigation of the storage stability of the samples (non-GLP), the test item proved to be stable under these storage conditions.
Vehicle:
yes
Remarks:
Swedish Standard Medium (SIS)
Details on test solutions:
Swedish Standard (SIS) medium was prepared according to the test guideline and was used for cultivation and testing of Lemna minor. Analytical grade salts were dissolved in sterile purified water to obtain the stock solutions. The pH was adjusted to 6.5 ± 0.2 with either 0.1 or 1 M HCl or NaOH and the volume adjusted to one litre with ultra-pure water. The medium was sterilized before use.
Test organisms (species):
Lemna minor
Details on test organisms:
TEST SYSTEM AND CULTURE CONDITIONS
- Common name: Exponential growing plant monoculture of Lemna minor.
- Culture: All-glass vessel containing test water and the exponentially growing plant monoculture.
- Cultivation: At least seven days before testing, sufficient colonies were transferred aseptically into fresh sterile medium and cultured for 7-10 days under the conditions of the test.
- Illumination: Continuous (6500–10000 lux) from Osram Fluora L18W77.
- Temperature: 24 ± 2 °C
- Control of sensitivity: Yearly, with 3,5-dichlorophenol. According to guideline ISO/CD 20079, the EC50 value based on the frond number’s growth rate should lie in the range 1.8–3.6 mg/l; this level of sensitivity was attained (latest data: March 2018).
Test type:
static
Water media type:
freshwater
Total exposure duration:
7 d
Test temperature:
24 ± 2 °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:
MEASURED CONCENTRATIONS
- At the beginning of the test: 523, 159, 49.3, 16.3 and 5.03 mg/l;
- At the end of the test: 513, 157, 47.0, 14.7 and 4.28 mg/l.
Details on test conditions:
TEST CONDITIONS
- Test vessel : 400 ml beakers, all-glass, with 200 ml of test water. The beakers were covered with black paper to ensure that illumination comes only from above and not from the sides.
- Pre-culture: exponentially growing plant monoculture of Lemna minor; healthy plants with 2-4 fronds were used; the culture was visibly free from contamination by other organisms such as algae and protozoa.
- Illumination: Intensity - continuous (6500–10000 lux) from Osram Fluora L18W77 and CH Lighting F18T8/6500K EUP. Homogeneity - within ±15%
- Incubation: Beakers were incubated on a black non-reflecting surface.

RANGE-FINDING STUDY
Prior to the definitive test a non-GLP range finding test with the nominal concentrations of 10, 100 and 500 mg/l of Acid Brown 396:1 (test item as a whole) was performed (3 replicates for the blank controls and 2 replicates per test concentration).
The percentage of inhibition for the different concentrations in this non-GLP range finding test, as well as the determined concentrations at the start and the end of the test. The photometry determinations indicate that the test item concentrations are stable. Static conditions were therefore applied in the definitive test.
Reference substance (positive control):
not specified
Remarks:
Blank control (Bn); containing test water and duckweed (six replicates) and pure test water as blank controls.
Key result
Duration:
7 d
Dose descriptor:
EC50
Remarks:
Frond number
Effect conc.:
> 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
EC50
Remarks:
Frond number
Effect conc.:
ca. 802 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
yield
Remarks on result:
other: extrapolated value
Key result
Duration:
7 d
Dose descriptor:
EC10
Remarks:
Frond number
Effect conc.:
ca. 258 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
EC10
Remarks:
Frond number
Effect conc.:
ca. 39.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Key result
Duration:
7 d
Dose descriptor:
LOEC
Remarks:
Frond number
Effect conc.:
ca. 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
LOEC
Remarks:
Frond number
Effect conc.:
ca. 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Key result
Duration:
7 d
Dose descriptor:
NOEC
Remarks:
Frond number
Effect conc.:
ca. 158 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
NOEC
Remarks:
Frond number
Effect conc.:
ca. 158 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Key result
Duration:
7 d
Dose descriptor:
EC50
Remarks:
Dry weight
Effect conc.:
> 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
EC50
Remarks:
Dry weight
Effect conc.:
ca. 653 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
yield
Remarks on result:
other: extrapolated value
Key result
Duration:
7 d
Dose descriptor:
EC10
Remarks:
Dry weight
Effect conc.:
ca. 136 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Dry weight
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
EC10
Remarks:
Dry weight
Effect conc.:
ca. 13.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Key result
Duration:
7 d
Dose descriptor:
LOEC
Remarks:
Dry weight
Effect conc.:
ca. 158 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
LOEC
Remarks:
Dry weight
Effect conc.:
ca. 158 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Key result
Duration:
7 d
Dose descriptor:
NOEC
Remarks:
Dry weight
Effect conc.:
ca. 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
NOEC
Remarks:
Dry weight
Effect conc.:
ca. 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Details on results:
EFFECTS ON FROND NUMBER
With respect to growth rate the following effects, as compared to the untreated controls, were observed the 16% at 500 mg/l. No significant effects were observed at the concentrations of 158, 50.0, 15.8 and 5.00 mg/l. Based on these data and the nominal concentrations, the 7-days EC50 with respect to the frond number’s growth rate was estimated to be >500 mg/l. The EC10 was 258 mg/l (95% confidence interval: 136–374 mg/l). These ECx were computed using a linear regression with a Probit link function and a maximum likelihood algorithm. The LOEC with respect to the average frond number’s growth rate was determined to be 500 mg/l according to a Williams’ multiple t-test (one-sided smaller, alpha = 0.05). The corresponding NOEC is therefore 158 mg/l.
With respect to yield the following effects as compared to the untreated controls were observed: 42% at 500 mg/l. No significant effects were observed at the concentrations of 158, 50.0, 15.8 and 5.00 mg/l. Based on these data and the nominal concentrations, the 7-days EC50 with respect to the frond number’s yield was extrapolated to be 802 mg/l (95% confidence limits: 427–>802 mg/l). The EC10 was 39.1 mg/l (95% confidence interval: 2.52–88.3 mg/l). These ECx were computed using a linear regression with a Probit link function and a maximum likelihood algorithm. The LOEC with respect to the average frond number’s yield was determined to be 500 mg/l according to a Williams’ multiple t-test (one-sided smaller, alpha = 0.05). The corresponding NOEC is therefore 158 mg/l.

EFFECTS ON DRY WEIGHT
With respect to growth rate the following effects as compared to the untreated controls were observed: 21% at 500 mg/l and 11% at 158 mg/l. No significant effects were observed at the concentrations of 50.0, 15.8 and 5.00 mg/l. Based on these data and the nominal concentration, the 7-days EC50 with respect to the dry weight’s growth rate was estimated to be >500 mg/l. The EC10 was 136 mg/l (95% confidence interval: 38.1–227 mg/l). These ECx were computed using a linear regression with a Probit link function and a maximum likelihood algorithm. The LOEC with respect to the average dry weight’s growth rate was determined to be 158 mg/l according to a Williams’ multiple t-test (one-sided smaller, alpha = 0.05). The corresponding NOEC is therefore 50.0 mg/l.
With respect to yield the following effects as compared to the untreated controls were observed: 50% at 500 mg/l and 30% at 158 mg/l. No significant effects were observed at the concentrations of 50.0, 15.8 and 5.00 mg/l. Based on these data and the nominal concentrations, the 7-days EC50 with respect to the dry weight’s yield was extrapolated to be 653 mg/l (95% confidence limits: 282 mg/l–not determined). The EC10 was 13.6 mg/l (95% confidence interval: not determined). These ECx were computed using a linear regression with a Probit link function and a maximum likelihood algorithm. The LOEC with respect to the average dry weight’s yield was determined to be 158 mg/l according to a Williams’ multiple t-test (one-sided smaller, alpha = 0.05). The corresponding NOEC is therefore 50.0 mg/l.

VALIDITY OF THE TEST
The test is considered valid, if the following criterion is fulfilled: the doubling time of frond number in the control must be less than 2.5 days (60 h). The achieved value is 1.6 days, so this validity criterion was met.
Validity criteria fulfilled:
yes
Conclusions:
The 7-day EC50(based on the frond number’s growth rate) of Acid Brown 396:1(EC no.947-779-0) on the duckweed Lemna minor is > 500 mg/l. This value is based on the nominal concentrations of the test item.
Executive summary:

The growth inhibitory effects of Acid Brown 396:1 to the duckweed Lemna minor were investigated according to test guideline OECD 221 under static conditions over a period of seven days.

The test item Acid Brown 396:1 is solid, soluble and has a purity of 100%. The test solutions were prepared by appropriate dilutions of a stock solution in test water. The nominal concentrations were 500, 158, 50.0, 15.8 and 5.00 mg/l. Three parallel test vessels were used for each concentration of the test item and six vessels for the controls. The test concentrations of Acid Brown 396:1 during the 7-days test period were determined by photometryat the beginning of the test, as well as after 2, 5 and 7 days of exposure. Since all concentrations were within 80–120% of the nominal value the effective concentrations ECxwere assessed based on the nominal concentrations of the test item. The results of the toxicity assessment of Acid Brown 396:1 to the duckweed Lemna minor are summarized in the following table, showing ECxvalues based on the nominal concentrations of the test item.

Summarized results  

Parameter (0-7 d)

Frond number

Dry weight

Growth rate
[mg/l]

Yield
[mg/l]

Growth rate
[mg/l]

Yield
[mg/l]

EC50

>500

802*

>500

653*

EC10

258

39.1

136

13.6

LOEC

500

500

158

158

NOEC

158

158

50

50

*extrapolated value

The 7-day EC50(based on the frond number’s growth rate) of Acid Brown 396:1 on the duckweed Lemna minor is > 500 mg/l. This value is based on the nominal concentrations of the test item.

The validity criterion was fulfilled.

 

Description of key information

The growth inhibitory effects of Acid Brown 396:1(EC no.947-779-0) to the duckweed Lemna minor were investigated according to test guideline OECD 221 under static conditions over a period of seven days.The test item Acid Brown 396:1 is solid, soluble and has a purity of 100%.The test solutions were prepared by appropriate dilutions of a stock solution in test water. The nominal concentrations were 500, 158, 50.0, 15.8 and 5.00 mg/l. Three parallel test vessels were used for each concentration of the test item and six vessels for the controls.

The test concentrations of Acid Brown 396:1 during the 7-day test period were determined by photometry at the beginning of the test, as well as after 2, 5 and 7 days of exposure. Since all concentrations were within 80–120% of the nominal value the effective concentrations ECx were assessed based on the nominal concentrations of the test item.The 7-day EC50 (based on the frond number’s growth rate) of Acid Brown 396:1 (EC no. 947-779-0) on the duckweed Lemna minor is >500 mg/l. The validity criterion was fulfilled.

Key value for chemical safety assessment

EC50 for freshwater plants:
500 mg/L
EC10 or NOEC for freshwater plants:
258 mg/L

Additional information

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