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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:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From July 25 to July 08, 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Justification for Read Across is detailed in the endpoint summary.
Qualifier:
according to guideline
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
adopted: 23 March 2006
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples were taken at 0 hours (initial value) from fresh test solution, after 2 and 5 days from fresh and aged test solution and after 7 days from aged test solution from control and all test item concentrations.
Vehicle:
no
Details on test solutions:
The necessary amount of test item for preparing the stock solution S1 was weighed and transferred to a volumetric flask. Test medium was added up to the bench mark and the solution was homogenised by shaking. Lower concentrations of the test item were prepared by dilution of the stock solution.
Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Common name: aquatic plant duckweed, Lemna gibba G3 (Alismatales: Araceae).
- Source: the plants used in this study were taken as aseptic clones from the cultivation of the testing facility. They were primarily cultured by Dr. Janet Slovin, Horticulture Crops Quality Laboratory, U.S. Department of Agriculture. BARC-West, Bldg. 050 HH-4, Beltsville, MD 20705, U.S.A.
- Method of cultivation: the plants were cultured in appropriate glass beakers under continuous illumination with OSRAM light tubes (L30/32-930 and L30/W72-965), at 6500 - 7000 lux. The temperature was 24 ± 2 °C. Before the test was started, plants were cultured under the same illumination and temperature conditions as those to be used for the toxicity test. Young, light-green plants of similar size and comprising 2 - 5 fronds were transferred onto fresh medium and cultured for ≥ 3 weeks prior to testing, with two further transfers onto fresh medium before initiating the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Test temperature:
23.1 – 24.6 °C
pH:
7.45 – 7.65
Nominal and measured concentrations:
0 (control), 0.0186, 0.0596, 0.191, 0.610, 1.95, 6.25 and 20.0 mg/l.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml glass beakers containing 150 ml test medium.
- Cells density: colonies consisting of 3-4 fronds were transferred from the inoculum culture into the test vessels to give a total of 12 fronds per vessel. The size of plants and fronds were similar in appearance in each test vessel.
- No. of fronds per vessel: 12 per test vessel.

TEST MEDIUM / WATER PARAMETERS
The medium used for the test was 20xAAP Medium for Lemna gibba (according to OECD 221 (2006)).
- Stock solution I: K2HPO4 21 mg/l
- Stock solution II: MgSO4·7H2O 290 mg/l
- Stock solution III: NaNO3 510 mg/l; CaCl2·2H2O 90 mg/l; MgCl2·6H2O 240 mg/l
- Stock solution IV: H3BO3 3.7 mg/l; MnCl2·4H2O 8.3 mg/l; ZnCl2 0.066 mg/l; CoCl2·6H2O 0.029 mg/l; CuCl2·2H2O 2.4 x 10^-4 mg/l; Na2MoO4·2H2O 0.145 mg/l; FeCl3·6H2O 3.2 mg/l; Na2EDTA·2H2O 6.0 mg/l.
- Stock solution V: NaHCO3 300 mg/l ( *direct weighing of 300 mg NaHCO3 into one litre test medium).

OTHER TEST CONDITIONS
- Light intensity (mean): ~ 6800 lux.
- Light tubes: OSRAM light tubes (L 30 W77 and L 30 W25).
- Exposure to light: continuously.

OBSERVATIONS
Frond numbers in each test vessel were determined at the start of the test. Frond numbers and the appearance of the colonies were checked on t: 0, 2, 5 and 7 days as well as any change in plant development, frond size, necrosis or mortality and additional observations of test media or other abnormalities.
The dry weight of the fronds was determined at the end of the test. A representative batch of six times 12 fronds from the culture used for the test was dried to determine the dry weight at the start of the study.

MEASUREMENTS
The test temperature was measured daily, the pH-value of the test solutions was measured at t = 0 d, 2 d, 5 d and 7 d in one replicate of fresh and aged test solutions of all concentrations. Light intensity was measured once at test start.
Reference substance (positive control):
no
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
12.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
10.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
On day 2 no observations were made up to 0.610 mg/l. At 1.95 mg/l slightly discoloured roots and partly broken roots were observed. At 6.25 and 20.0 mg/l smaller offshoots and slightly discoloured, partly broken roots were observed.
On day 5 and 7 slightly discoloured, shorter roots and curved fronds were observed at 0.610 mg/l. At 1.95 mg/l small, pale offshoots and shorter, reddish
roots were observed. At the highest test item concentration level partly broken roots, small offshoots and partly red fronds were observed.

Significant inhibitory effects were determined for all parameters at 1.64, 5.25 and 18.4 mg/l (actual). The overall LOEC was determined to be 1.64 mg/l (actual), the corresponding NOEC at 0.397 mg/l (actual).
The actual EC10-value for yield of frond numbers was determined to be 0.297 mg/l, the actual EC10-value for growth rate of frond numbers was determined to be 0.837 mg/l. The actual EC10-value for yield of dry weight was determined to be 0.591 mg/l, the actual EC10-value for growth rate of dry weight was determined to be 1.10 mg/l.
The actual EC20-value for yield of frond numbers was determined to be 0.656 mg/l, the actual EC20-value for growth rate of frond numbers was determined to be 2.44 mg/l. The actual EC20-value for yield of dry weight was determined to be 1.09 mg/l, the actual EC20-value for growth rate of dry weight was determined to be 2.67 mg/l.
The actual EC50-value for yield of frond numbers was determined to be 2.54 mg/l, the actual EC50-value for growth rate of frond numbers was determined to be 12.3 mg/l. The actual EC50-value for yield of dry weight was determined to be 3.48 mg/l, the actual EC50-value for growth rate of dry weight was determined to be 10.1 mg/l.
Reported statistics and error estimates:
The statistical evaluation for day 7 was performed for yield of frond numbers, growth rate of frond numbers, growth rate dry weight and yield of dry weight. For the determination of endpoints based on actual concentrations the two lowest test item concentrations of 0.0186 and 0.0596 mg/L were not considered, as theses concentration levels were not analysed. A test for normality of the data was performed by calculating the Shapiro-Wilk’s statistic, a test for homogeneity of the data was performed according to Levene. The NOEC and LOEC were determined by using a multiple comparison method (Dunnett`s t-test). The EC10, 20- and EC50-values for yield of frond number, growth rate of frond number, yield of dry weight and growth rate of dry weight were determined by probit analysis following normal, logistic or Gompertz procedure. The evaluation of data was performed by SAS® (2002-2010).

Toxicological results

Test item actual*[mg/l]
Endpoint EC10 EC20 EC50 NOEC4) LOEC4)
Yield of frond numbers 0.2972) 0.6562) 2.542) 0.397 1.64
Growth rate of frond numbers 0.8373) 2.443) 12.33) 0.397 1.64
Yield of dry weight 0.5911) 1.091) 3.481) 0.397 1.64
Growth rate of dry weight 1.103) 2.673) 10.13) 0.397 1.64

Test item nominal [mg/l]
Endpoint EC10 EC20 EC50 NOEC4) LOEC4)
Yield of frond numbers 0.3722) 0.8042) 3.002) 0.61 1.95
Growth rate of frond numbers 1.053) 2.933) 13.83) 0.61 1.95
Yield of dry weight 0.7371) 1.331) 4.091) 0.61 1.95
Growth rate of dry weight 1.383) 3.203) 11.43) 0.61 1.95

1) Probit analysis following normal distribution

2) Probit analysis following logistic distribution

3) Probit analysis following Gompertz distribution

4) following Dunnetts-t-test

* based on the geometric mean of the analysed concentration levels

Determined concentrations

Sampling Test item found
mg/l % of nominal Geometric mean % Actual concentration based on geometric mean
Control 0 d n.d. - - -
2 d aged n.d. -
2 d fresh n.d. -
5 d aged n.d. -
5 d fresh n.d.  -
0.191 0 d < LOQ1) 45 45 0.086
2 d aged < LOQ1) 45
2 d fresh < LOQ1) 45
5 d aged < LOQ1) 45
5 d fresh < LOQ1) 45
0.61 0 d 0.474 78 65 0.397
2 d aged 0.42 69
2 d fresh 0.464 76
5 d aged 0.263 43
5 d fresh 0.492 81
1.95 0 d 1.76 90 84 1.64
2 d aged 1.53 78
2 d fresh 1.78 91
5 d aged 1.49 76
5 d fresh 1.77 91
6.25 0 d 5.64 90 84 5.25
2 d aged 4.8 76
2 d fresh 5.88 93
5 d aged 4.94 78
5 d fresh 5.64 90
20 0 d 18.3 92 92 18.4
2 d aged 18 90
2 d fresh 18.5 93
5 d aged 19 95
5 d fresh 18.4 92

- = not calculated; n.d. = not detectable; LOQ = 0.170 mg/l test item; LOD = 0.0510 mg/l test item; 1) set to LOQ/2 for calculation of actual concentrations according to recommendations of OECD 23 “Guidance Document on Aquatic toxicity testing of difficult substances and mixtures”

The initial mean measured concentration of the test item in the test item solutions was 77 % of nominal. The overall mean concentration of fresh and aged samples was 72 % of nominal.

Validity criteria fulfilled:
yes
Remarks:
the test is valid as the doubling time of control frond numbers was calculated to be 37.9 h, corresponding to 1.581 days at day 7. This is less than the maximum allowable doubling time set in OECD guideline 221 (60 h, 2.5 days)
Conclusions:
ECr50 (7d) frond number: 12.3 mg/l (geom mean)
ECr50 (7d) dry weight: 10.1 mg/l (geom mean)
ECb50 (7d) frond number: 2.54 mg/l (geom mean)
ECb50 (7d) dry weight: 3.48 mg/l (geom mean)
Executive summary:

Method

The inhibitory effects based on the average specific growth rate and the yield (absolute increase in plant size) of the test item to the duckweed Lemna gibba was investigated over a period of 7 days. The semi-static test was performed at concentrations of 0.0186, 0.0596, 0.191, 0.610, 1.95, 6.25 and 20.0 mg/l of the active ingredient. The initial mean measured concentration of the test substance in the test item solutions was 77 % of nominal. The overall mean concentration of fresh and aged samples was 72 % of nominal. Toxicological endpoints were evaluated using nominal concentrations of the test item and actual concentrations based on the geometric mean concentration of each test item concentration level.

Results

Significant inhibitory effects were determined for all parameters at 1.64, 5.25 and 18.4 mg/l (actual). The overall NOEC at 0.397 mg/l (actual).

The actual EC50-value for yield of frond numbers was determined to be 2.54 mg/l, the actual EC50-value for growth rate of frond numbers was determined to be 12.3 mg/l. The actual EC50-value for yield of dry weight was determined to be 3.48 mg/l, the actual EC50-value for growth rate of dry weight was determined to be 10.1 mg/l. The test is valid as the doubling time of control frond numbers was calculated to be 37.9 h, corresponding to 1.581 days at day 7. This is less than the maximum allowable doubling time set in OECD guideline 221 (60 h, 2.5 days).

 

Description of key information

ECr50 (7d) frond number: 12.3 mg/l (geom mean)

ECr50 (7d) dry weight: 10.1 mg/l (geom mean)

Key value for chemical safety assessment

EC50 for freshwater plants:
10.1 mg/L
EC10 or NOEC for freshwater plants:
0.397 mg/L

Additional information

In order to assess the potential toxicity to aquatic plants of the Acid Red 357 (AR357) the available data on the structural analogue Acid Red 315 (AR315 - Similar Substance 01) were considered.

The inhibitory effects based on the average specific growth rate and the yield (absolute increase in plant size) of the AR315 to the duckweed Lemna gibba was investigated over a period of 7 days. The semi-static test was performed at concentrations of 0.0186, 0.0596, 0.191, 0.610, 1.95, 6.25 and 20.0 mg/l of the active ingredient. The overall mean concentration of fresh and aged samples was 72 % of nominal.

Significant inhibitory effects were determined for all parameters at 1.95, 6.25 and 20.0 mg/l (nominal) and 1.64, 5.25 and 18.4 mg/l (actual). The overall NOEC at 0.610 mg/l (nominal) and 0.397 mg/l (actual).

The actual EC50-value for yield of frond numbers was determined to be 2.54 mg/l, the actual EC50-value for growth rate of frond numbers was determined to be 12.3 mg/l. The actual EC50-value for yield of dry weight was determined to be 3.48 mg/l, the actual EC50-value for growth rate of dry weight was determined to be 10.1 mg/l. The test is valid as the doubling time of control frond numbers was calculated to be 37.9 h, corresponding to 1.581 days at day 7. This is less than the maximum allowable doubling time set in OECD guideline 221 (60 h, 2.5 days).

 

As above mentioned, the test was conducted on the structural analogue AR315, which shares with AR357 the same structure, except for the fact that AR315 is a mono-sulphonated disodium salts, while AR357 is a di-sulphonated trisodium salt. It is expected that this difference has no impact on the aquatic plants toxicity potential, thus the read across approach proposed can be considered reliable and the tests outcomes can be considered as suitable in order to assess the AR357 behaviour.

AR357 and AR315 are both textile, pre-metalized complex dyes 1:2 and they are manufactured for the same use, thus the characteristics of the final product are strictly similar and share the same functionality. The typical commercial batches present some differences. The main component AR357 is commonly greater than 55 percent, while in the case of AR315 the main component was stated at 80 %. From this point of view, the major active ingredient concentration of AR315 leads to consider the read-across approach as conservative.

The major difference in the composition is due to the elevated concentration of inorganic salts (i.e. sodium chloride and disodium sulphate) in the AR357, which is a consequence of the salification procedure. In general, the impurities do not impact the experimental results and the endpoint assessment.

Considering that the substances are both salts derived from strong acids (sulphonic acid derivatives), it is expected that they would be completely dissociated and stable in water for the whole test period in that form.