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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:
2017-07-03 to 2017¬07-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
2006
Deviations:
yes
Remarks:
substance tested with its Water Soluble Fraction
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- CAS no: 61419-02-1
- Batch no.: PO 4924335980 - ML 2005975931
Analytical monitoring:
yes
Details on sampling:
- Sampling method: The samples for the analysis of the test item from the freshly prepared media were taken out directly from the test solutions before distribution into the replicates. The samples for the chemical analysis from the old media were taken from the pooled test replicates per concentration and control. When taking samples it was ensured that the finely dispersed particles were analyzed as well. Samples were taken and homogenized directly before the measurement to avoid losses by precipitation. Since the separation of finely dispersed particles was not possible for this test item in the preliminary tests, care was taken to make sure that any undissolved part in the test solutions would be correctly represented by the analytical values in the definitive test.

- Sample storage conditions before analysis: All original samples were stored at room temperature. Prepared samples were stored at room temperature in an autosampler until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Since the test item is a mixture and in view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD Series No. 23 (2000)), is to expose the organisms to a Water Accommodated Fraction (WAF) / Water Soluble Fraction (WSF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents (as defined in the guidelines mentioned above).
- Growth medium: 20X-AAP-medium (Algal Assay Procedure Medium), pH-value 7.5 ± 0.1
- Method: Water Soluble Fraction (WSF); test solutions were prepared with deionized water and the saturated solutions were treated as specified in section 4.2. to obtain test solutions in test medium.
- Differential loading: 0.632, 2.00, 6.32, 20.0, 63.2, 200 mg/L
- Controls: Negative control (without test item), positive control (with reference item 3,5-Dichlorophenol)
- Evidence of undissolved material: No particles or precipitates were observed in the WSFs of any of the test loadings.
Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Common name: Duckweed,
- Strain: Lemna gibba G3
- Source: EUROFINS-GAB GMBH, Eutinger Str. 24, D-75223 Niefern-Oschelbronn, Germany
- Method of cultivation: The species was bred in the test facility. Density was kept sufficiently low to prevent conglomerates of plants on the surface. At least once per week, plants were transferred to freshly prepared growth medium. Growth media and breeding vessels were autoclaved before use to enable the breeding of axenic cultures.

ACCLIMATION
- Acclimation period: Yes, 7 days
- Acclimation conditions: Under test conditions
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Remarks on exposure duration:
according to guideline
Post exposure observation period:
no
Hardness:
n/a
Test temperature:
23.1 - 25.3 °C; mean value: 24.6 °C
pH:
Test concentrations: 7.40 - 8.35 (fresh medium), 7.41 - 8.52 (old medium)
Negative control: 7.46 - 8.35 (fresh medium), 7.46 - 8.50 (old medium)
Dissolved oxygen:
n/a
Salinity:
n/a
Conductivity:
n/a
Nominal and measured concentrations:
Nominal: 0.632, 2.00, 6.32, 20.0, 63.0, 200 mg/L
Measured: 0.0209 to 0.0578 mg/L
Details on test conditions:
TEST SYSTEM
- Incubation chamber used: Yes
- Test vessel: Dishes
- Type: Closed
- Material, size, headspace, fill volume: Crystallizing dishes of glass, vol. 900 mL, filled with ca. 500 mL growth medium
- Type of cover: Glass tops
- Aeration: No
- Agitation: No
- Renewal rate of test solution: daily
- No. of colonies per vessel: 4 uniform
- No. of fronds per colony: 3
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: Yes

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Continuous fluorescent light
- Light intensity and quality: 1100 - 444o lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: Manual counting
- Determination of biomass: Dry weight

RANGE-FINDING STUDY
- Test concentrations: 10, 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
7 d
Dose descriptor:
EL10
Effect conc.:
> 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EL20
Effect conc.:
> 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
EL50
Effect conc.:
> 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
other: NOEL
Effect conc.:
200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
other: LOEL
Effect conc.:
> 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EL10
Effect conc.:
71.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EL20
Effect conc.:
> 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EL50
Effect conc.:
> 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
other: NOEL
Effect conc.:
200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
other: LOEL
Effect conc.:
> 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Any visual signs of phytotoxicity (abnormalities):
- Decrease in frond size: No
- Necrosis / chlorosis: No
- Sinking of fronds: No
- Any stimulation of growth found in any treatment: No
- Effect concentrations exceeding solubility of substance in test medium: No

VALIDITY OF THE TEST
Doubling times of the frond numbers after 7 days: 1.86 days (45 hours) corresponding to an average specific growth rate of 0.374 [d_1] (14-fold increase). The test is valid.

TEST CONCENTRATIONS: With decreasing loadings the measured concentrations showed a general tendency of decrease, which was however not proportional to the loading. At the end of the 24 hour exposure intervals on days 1, 2 and 7 the measured values were < LOQ with one exception.
Results with reference substance (positive control):
Growth rate inhibition (number of fronds): ErC50 = 6.95 (95% confidence interval: 5.75-8.00)
Yield inhibition (number of fronds): EyC50 = 4.35 (95% confidence interval: 3.88-4.91)
Growth rate inhibition (dry weight): ErdwC50 = 6.16 (95% confidence interval: 3.86-4.85)
Yield inhibition (dry weight): EydwC50 = 4.31 (95% confidence interval: 3.86-4.85)
Reported statistics and error estimates:
NOEL/LOEL was determined by calculation of the statistical significance of inhibition of growth rates and yield in comparison to the control: One Way Analysis of Variance (ANOVA) and DUNNETT'S test were used as a 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 a-value (acceptable probability of incorrectly concluding that there is a difference) was oc = 0.05.

EL10-values (0 - 7 d) of the yield (frond number and dry weight) inhibition were calculated by sigmoidal dose-response regression. Calculations of the confidence intervals of EL10-values were carried out from the best fit values, the standard error and the t-distribution with the software GraphPad Prism. For details, see section 14. The EL10-, EL20- and EL50-values of growth rate (frond number and dry weight) inhibition were derived directly from the observations. Also the EL20- and EL50-values of the yield (frond number and dry weight) inhibition were derived directly from the observations.

Calculations were carried out using the following software:
- Excel, MICROSOFT CORPORATION
- SigmaPlot, SPSS INC.
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Table 1: Measured Exposure Concentrations and Percent of the Test Item during the Definitive Test;

Determination of the test item via LC-MS/MS

Sampling date

2017-07-05 Fresh media (day 0)

2017-07-06 Old media (day 1)

2017-07-06 Fresh media (dayl)

2017-07-07 Old media (day 2)

2017-07-11 Fresh media (day 6)

2017-07-12 Old media (day 7)

Start of analysis

2017-07-05

2017-07-06

2017-07-06

2017-07-07

2017-07-11

2017-07-12

Nominal loading of the test item in the WSF [mg/L]

Test item

Meas. cone. [mg/L]

Meas. cone. [mg/L]

Meas. cone. [mg/L]

Meas. cone. [mg/L]

Meas. cone. [mg/L]

Meas. cone. [mg/L]

200

0.05781>

0.00463

0.0209

< LOQ

0.0336

< LOQ

63.0

0.0307

< LOQ

0.0414

< LOQ

0.0317

< LOQ

20.0

0.0369

< LOQ

0.0392

< LOQ

0.0207

< LOQ

6.32

0.0302

< LOQ

0.0319

< LOQ

0.0157

< LOQ

2.00

0.0174

< LOQ

0.0221

< LOQ

0.00926

< LOQ

0.632

0.00825

< LOQ

0.00332

< LOQ

0.0118

< LOQ

Control

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

Meas. Cone. = Measured concentration of the test item, dilution factors taken into account, mean value

of 2 injections, at 2017-07-06 single injection

LOQ = Limit of quantification (3.00 ug test item/L)

1) = Reanalyzed on 2017-07-06, single determination

Table 2: Frond Numbers

Nominal Test Item Loading [mg/L] Repl. no. Frond numers per study
0 days 2 days 5 days 7 days
200 1 12 19 61 133
2 12 26 72 150
3 12 23 67 146
Mean 12 23 67 143
63.2 1 12 22 69 143
2 12 20 68 145
3 12 21 72 165
Mean 12 21 70 151
20.0 1 12 20 72 148
2 12 25 70 169
3 12 21 78 146
Mean 12 22 73 154
6.32 1 12 21 72 168
2 12 26 68 168
3 12 20 73 156
Mean 12 22 71 164
2.00 1 12 27 79 173
2 12 20 72 159
3 12 22 71 152
Mean 12 23 74 161
0.632 1 12 25 78 160
2 12 21 75 166
3 12 20 71 166
Mean 12 22 75 164
Control 1 12 19 65 159
2 12 23 73 172
3 12 21 73 153
4 12 25 80 165
5 12 22 73 179
6 12 20 74 158
Mean 12 22 73 164
Validity criteria fulfilled:
yes

Description of key information

The toxicity of the read across substance Reaction products of γ-butyrolactone, naphthalene-1,6-diol and phthalic anhydride (EC 946-798-2) to aquatic plants was assessed according to OECD guideline 221 under GLP conditions. The 7-day ErL10 and ErL50 was determined to be > 200 mg/L.

Key value for chemical safety assessment

Additional information

No data on toxicity to aquatic plants other than algae were available for the substance itself. Therefore, a read across to the close structural analogue Reaction products ofγ-butyrolactone, naphthalene-1,6-diol and phthalic anhydride(EC 946-798-2) was conducted.

Key study

The effects of a Water Soluble Fraction (WSF) of the close structural analogue (EC 946-798-2) on the growth of the monocotyledonous aquatic plant species Lemna gibba was determined according to OECD Guideline 221 and in compliance with GLP principles (BASF SE, 2017). Since the test item is poorly water soluble, a modification of the standard method for the preparation of aqueous media was performed and a WSF in accordance with ECETOC 1996, OECD Series No. 23 (2000) prepared. Furthermore, as the test item contains a large proportion of unidentified by-components (UVCB substance), it is treated as a mixture and the results are based on nominal loadings rather than on mean measured concentrations. Lemna gibba was exposed to the test item for 7 days under semi-static conditions with daily renewal. Based on a preliminary test, 6 nominal test item concentrations were tested in a geometrical series with a dilution factor of 2nd root of 10: 0.632, 2.00, 6.32, 20.0, 63.2 and 200 mg/L. Three replicates were investigated for each test concentration and six for the control. Frond numbers were assessed on days 0, 2, 5 and 7. Environmental parameters (light, pH and temperature) were within the acceptable limits. The measured concentrations (analyzed via LC-MS/MS) of the test item in fresh media on days 0, 1 and 6 in the highest tested loading were in the range of 0.0209 to 0.0578 mg test item/L. As a result, the 7-day ErL10, ErL20 and ErL50 as well as the LOEL for the growth rate Inhibition was determined to be > 200 mg/L. The 7-day NOEL (growth rate) was established to be 200 mg/L. For the inhibition of yield, the 7-day EyL2, EyL50 and LOEL was determined to be > 200 mg/L. The 7-day EyL10 and NOEL (inhibition of yield) was established to be 71.8 (15.6 - > 200) mg/L and 200 mg/L, respectively. All validity criteria of the test guideline were fulfilled.