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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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
The read across approach is detailed into the document attached to the IUCLID section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples of all fresh test item concentrations and the control at exposure initiation and all old test item concentrations obtained at the first renewal were chemically analyzed. However, the test item conentration of 0.064 and 0.013 mg/l are below LOD (LOD = 0.1 mg/l). Moreover, samples of fresh test item concentrations of 1000 and 0.32 mg/l at each renewal as well as old test item concentrations of 1000 and 0.32 mg/l at the second renewal and at exposure termination were chemically analyzed.
Details on test solutions:
TEST SOLUTIONS
The appropriate amount of the test item was weighed into a glass beaker and quantitatively transferred to volumetric and filled up to 1000 ml with 20X AAP medium. The test item concentration of 1000 mg/l was visually hetrogeneous, therefrore the contant of flask was sonicated for 5 minutes. Then, the test item concentration of 1000 mg/l ws visually homogeneous. Lower test item concentrations were prepared by sequential dilutions with the 20X AAP medium in a ratio 1:4 (v/v) (i.e. 200 ml of higher test item concentration was mixed with 800 ml of the 20X AAP medium).
Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Common name: duckweed
- Origin: Canadian Phycological culture Centre (CPCC), Department of Biology, University of Waterloo, Ontario, Canada.
- Cultivation at test facility: duckweed was transferred from agar bevels to the fresh 20X AAP medium in glass beakers with a capacity of 600 ml with transparent lids and incubated in 22 - 26 °C with constant illumination (pre-culturing). The duckweed culture was inoculated to fresh medium once a week.

ACCLIMATION
- Acclimation period: a pre-culture was started seven days before exposure.
- Organisms selection: only organisms in good physiological condition without any discoloration were used for the inoculation of the test item concentrations and the control.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Test temperature:
24.5 - 25.0 °C
pH:
Start of the test: 7.43 - 7.61
End of the test: 8.02 - 9.05
During test: 7.35 - 8.76
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 0.013, 0.064, 0.32, 1.6, 8.0, 40, 200 and 1000 mg/l (test range based on a preliminary test).
Details on test conditions:
TEST SYSTEM
- Test vessel: glass crystallizers of 7 cm diameter and 4 cm depth were used as test vessels in order to provide enough space for roots and to prevent plants from overlapping at exposure termination.
- Fill volume: each test vessel contained 150 ml of a given test item concentration or control.
- Type of cover: transparent lids were used to minimize evaporation and accidental contamination, allowing necessary air exchange.
- Renewal rate of test solution: two renewals, according to the stability test results.
- No. of colonies per vessel: at exposure initiation 3 colonies.
- No. of fronds per colony: 3 fronds of similar size; each replicate was inoculated with a total of 9 fronds.
- No. of vessels per concentration: 3 replicates.
- No. of vessels per control: 6 replicates.

GROWTH MEDIUM
- Standard medium used: the 20X AAP medium recommended by the OECD Guideline No. 221 (2006) was the culturing medium for test organism and the diluent/solvent for the test item.
- Preparatyion: the 20X AAP medium was prepared on the basis of deionised water by adding stock solutions of reagent-grade chemicals. The medium was sterilised by autoclaving before addition of the stock solution of sodium hydrogen carbonate – the buffering ingredient..
- Adjustment of pH: after 24 h of aeration the pH value of test medium was measured and adjusted with 0.1 M HCl, if necessary.
- Stock solution: the stock solutions were renewed on regular basis for Lemna gibba culturing and stored in a refrigerator.
- Alkalinity: 292 mg/l as NaHCO3.
- Hardness: 296 mg/l as CaCO3.

TEST CONDITIONS
- Constant conditions: the constant conditions throughout exposure were provided with the thermostatic chamber. The temperature was continuously recorded by electronic device with a sensor submerged in an additional test vessel with 150 ml of the 20X AAP medium.
- Photoperiod: the tests were conducted with constant illumination, using fluorescent light source (six 24 W light bulbs).
- Light intensity and quality: the light intensity was measured at exposure initiation, twice during exposure and at exposure termination with 2π receptor lux-meter (Sonopan L-50, Poland).

EFFECT PARAMETERS MEASURED
In order to quantify the test item related effects on vegetative growth, the number of fronds in each replicate was counted. The total number of fronds in each test vessel was counted on days 2 and 5 and at exposure termination in the first preliminary test and on days 3 and 5 and at exposure termination in the second preliminary test and definitive test. Only visibly distinct fronds were counted. At the same time observations of plant development were performed: frond size, shape and appearance (necrosis, chlorosis, gibbosity or bending of fronds), colony break-up or loss of buoyancy, root length and appearance. Growth of plant cultures in the test item concentrations was compared with that of the control.

The dry weight of the representative sample of the duckweed culture used as the inoculum was measured at exposure initiation. The dry weight of all plants from each test vessel was measured after exposure termination. All colonies (with roots) were transferred onto previously weighed microscopic slides and dried at 60 °C in a laboratory oven until the constant weight.

MEASUREMENTS
- pH: the pH values were measured in fresh test item concentrations and the control before split up into replicates at exposure initiation and at each renewal. The pH values were also measured in old test item concentrations and the control at each renewal and at exposure termination in pooled replicates.

TEST WITH REFERENCE SUBSTANCE:
- Test item: reference substance, 3,5-dichlorophenol.
- Test type: semi-static conditions with two renewals.
- Duration: 7 day exposure.
- Concentrations: 5 concentrations of the reference substance ranging from 0.32 to 32 mg/l were used.
- No. of vessels per concentration: 3 replicates.
- No. of vessels per control: 6 replicates.

VALIDITY CRITERIA:
- The doubling time of frond number in the control should be less than 2.5 days.
- The average specific growth rate in the control between day 0 and day 7 should be higher than 0.275 per day.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorphenol
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
based on frond number and based on dry weight
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
(yield) based on frond number
Details on results:
ErC10 (7d) > 1000 mg/l (nominal), based on frond number
NOErC (7d): 40 mg/l (nominal), based on frond number
ErC10 (7d): 36.26 mg/l (nominal), based on dry weight (95 % C.I.: 11.90 - 140.01 mg/l)
NOErC (7d): 1.6 mg/l (nominal), based on dry weight

EyC10 (7d): 113.66 mg/l (nominal), based on frond number (95 % C.I.: 0.80 - 277.93 mg/l)
NOEyC (7d): 40 mg/l (nominal), based on frond number
ErC50 (7d): 675.88 mg/l (nominal), based on dry weight (95 % C.I.: 430.24 - 1240.80 mg/l)
ErC10 (7d): 7.28 mg/l (nominal), based on dry weight (95 % C.I.: 2.43 - 14.90 mg/l)
NOEyC (7d): 1.6 mg/l (nominal), based on dry weight

VISUAL OBSERVATIONS
On days 3, 5 of exposure and at exposure termination in the test item concentrations of 0.013, 0.064, 0.32 and 1.6 mg/l no distinctive changes from the normal development of plants in the control were observed. In the test item concentrations of 8.0, 40 and 200 mg/l, coloured roots were observed. In the test item concentration of 1000 mg/l, coloured roots and frond were observed.

MEASURED CONCENTRATIONS
Measured concentrations: in test media, both before and after renewals, measured concentrations were in the range of 88.0 - 101.1% of nominal concentrations (0 (control), 0.064 and 0.013 mg/l solutions were < LoQ).
Results with reference substance (positive control):
Based on frond number:
7-d ErC50: 11.9 mg/l
7-d ErC10: 8.0 mg/l
7-d EyC50: 9.5 mg/l
7-d EyC10: 4.0 mg/l
Reported statistics and error estimates:
For the determination of NOEC, LOEC and EC-values, three replicates were included for the test concentrations and six replicates for the control. EC-values of the growth rate and yield inhibition were estimated empirically based on the results of the definitive test. NOEC/LOEC were determined by calculation of the statistical significance of inhibition of growth rates and yield in comparison to the control using One Way Analysis of Variance (ANOVA) and Dunnett's test as standard. A normality test and an equal variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. P-values for both Normality and Equal Variance test were 0.05. The α-value (acceptable probability of incorrectly concluding that there is a difference} was α=0.05. The normality test failed for the calculation of growth rate and yield (dry weighed). To pass the normality test, the data of yield were transformed. For the calculation of the growth rate, no transformation data was found to pass the normality test.

Inhibition of growth rate and yield at exposure termination (day 7)

Nominal test item conc. (mg/l) % inhibition
based on fond number
% inhibition
based on dry weight
growth rate yield growth rate yield
0 (control) 0.0 1.1 0.0 0.0
0.013 -3.8 -12.8 -5.1 -19.3
0.064 0.0 0.4 -3.4 -11.6
0.32 3.2 9.6 -1.7 -5.3
1.6 0.3 1.2 -1.7 -5.5
8.0 2.5 7.6 4.8 15.9
40 0.8 2.6 5.9 19.2
200 4.4 12.0 12.9 37.3
1000 12.1 32.1 21.6 54.1
Validity criteria fulfilled:
yes
Remarks:
doubling time of frond number in the control was 1.6 days (the factor of frond number in the control between 0 and 7 day was 19.5) and the average specific growth rate in the control between day 0 and day 7 was 0.424 per day
Conclusions:
ErC50 (7d) > 1000 mg/l (nominal), based on frond number and based on dry weight
EyC50 (7d) > 1000 mg/l (nominal), based on frond number
Executive summary:

The effect of test item was assessed in duckweed, Lemna gibba, over an exposure period of 7 days, in semi-static test. The experiments was conducted following the testing procedures outlined into the OECD guideline 221. Based on the results of two non-GLP preliminary tests, the nominal test item concentrations included in the study were 0 (control), 0.013, 0.064, 0.32, 1.6, 8.0, 40, 200 and 1000 mg/l. In test media, both before and after renewals, measured concentrations were in the range of 88.0 - 101.1 % of nominal concentrations (0 (control), 0.064 and 0.013 mg/l solutions were < LoQ). All validity criteria were met and therefore the study can be considered as valid.

The 7-d ErC50, based on frond number, was determined to be higher than 1000 mg/l, as well as the 7-d ErC50, based on dry weight. On the basis of the growth rate, the effects observed at 1000 mg/l regard the 12 and 32 % of population, based on frond number and dry weight, respectively.

On the basis of the yield, the 7-d EyC50, based on frond number, resulted to be higher than 1000 mg/l; the 7-d EyC50, based on dry weight, resulted to be 675.88 mg/l (95% C.I.: 430.24 - 1240.80 mg/l).

Conclusion

ErC50 (7d) > 1000 mg/l (nominal), based on frond number and based on dry weight

EyC50 (7d) > 1000 mg/l (nominal), based on frond number

Description of key information

ErC50 (7d) > 1000 mg/l (nominal), based on frond number and based on dry weight

EyC50 (7d) > 1000 mg/l (nominal), based on frond number

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:
1 000 mg/L

Additional information

There are no information about the potential toxicity to aquatic plants of Direct Orange 118, thus the available data on structural analogous Similar Substance 01 have been taken into consideration. The read across approach can be considered as reliable and suitable for the purpose; details and explanations are detailed in the report attached to the IUCLID section 13.

The effect of Similar substance 01 was assessed in duckweed, Lemna gibba, over an exposure period of 7 days, in semi-static test. The experiments was conducted following the testing procedures outlined into the OECD guideline 221.

In test media, both before and after renewals, measured concentrations were in the range of 88.0 - 101.1 % of nominal concentrations. The 7-d ErC50, based on frond number, was determined to be higher than 1000 mg/l, as well as the 7-d ErC50, based on dry weight. On the basis of the growth rate, the effects observed at 1000 mg/l regard the 12 and 32 % of population, based on frond number and dry weight, respectively.

On the basis of the yield, the 7-d EyC50, based on frond number, resulted to be higher than 1000 mg/l; the 7-d EyC50, based on dry weight, resulted to be 675.88 mg/l (95% C.I.: 430.24 - 1240.80 mg/l). The effects observed at 1000 mg/l regard the 22 and 54 % of population, based on yield frond number and dry weight, respectively.