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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
Study period:
June from 04th to 14th, 2014
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:
Justification for read across approach is detailed in the report attached to the IUCLID section 13
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
23 March 2006
Deviations:
yes
Remarks:
no major deviations to the guideline (pH of the control increased by more than 1.5 units on day 5. The test is still valid, as it was shown that validity criteria are met).
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples were taken at 0 hours (initial value) from fresh test solution, after 3 and 5 days from fresh and aged test solution and after 7 days from aged test solution from control and all test item concentrations.
Nominal test item concentrations and control were analysed at t=0d from fresh test solutions and at t=3d and 5d from fresh and aged test solutions.
Details on test solutions:
The necessary amount of test item for preparing the stock solution 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 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.
- Source: the plants used 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: plants were cultured in appropriate glass beakers under continuous illumination with OSRAM light tubes at 6500 - 7000 lux. The temperature was 24 ± 2 °C.

ACCLIMATION
- Culturing media and conditions: before the test was started, plants were cultured under the same illumination and temperature conditions as those to be used for the toxicity test.
- Preparation for 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:
22.0 – 24.7 °C
pH:
7.47 – 7.64 of fresh solutions
Nominal and measured concentrations:
0 (control), 0.954, 3.05, 9.77, 31.3 and 100 mg/l (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml glass beakers each containing 150 ml test medium to allow the plants to grow without touching the sides of the glass vessels.
- Renewal rate of test solution: colonies were transferred two times to new test solutions (days 3 and 5).
- Control end cells density: colonies consisting of 3-4 fronds were transferred from the inoculum culture into the test vessels.
- No. of colonies per vessel: 12 fronds per vessel. The size of plants and fronds were similar in appearance in each test vessel.
- No. of vessels per concentration: one replicate.
- No. of vessels per control: one replicate.

GROWTH MEDIUM
- Standard medium used: the medium used for the test was 20xAAP Medium for Lemna gibba (according to OECD 221 (2006)).

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

EFFECT PARAMETERS MEASURED
- Determination of dry weight: at the end of the test all the fronds from each test vessel were dried at 60 °C for about 72 hours. A representative batch of 6 times 12 fronds from the culture used for the test was dried to determine the dry weight at the start of the study.
- Determination of growth rate: means of frond number for each test concentration at each observation time were calculated. Means of dry weight for each test concentration at day 7 were calculated. The specific growth rate for frond number and dry weight was calculated as the logarithmic increase for each replicate for control and treatments.
- Determination of yield: effects on yield are determined on the basis of total frond numbers and dry weight in each vessel at the start and end of the test.

MEASUREMENTS
- temperature: the test temperature was measured daily.
- pH-value: measured at t = 0 d, 3 d, 5 d and 7 d in one replicate of fresh and aged test solutions of all concentrations.
- Light intensity: measured once at test start.
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
69.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks:
yield
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks:
growth rate
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
50.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield of dry weight
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
79.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
of dry weight
Details on results:
Yield of frond numbers NOEC (7d): 31.3 mg/l (nominal) (following Dunnetts-t-test)
Growth rate of frond numbers NOEC (7d): 31.3 mg/l (nominal) (following Dunnetts-t-test)
Yield of dry weight NOEC (7d): 9.77 mg/l (nominal) (following Jonckheere-Terpstra)
Growth rate of dry weight NOEC (7d): 9.77 mg/l (nominal) (following Jonckheere-Terpstra)

Significant inhibitory effects were determined for yield of frond numbers and for growth rate of frond numbers at 100 mg/l (nominal).
On day 3 slightly shorter roots were observed at 31.3 mg/l. At 100 mg/l the fronds were curved and coloured red and offshoots were smaller. The roots were shortened. On day 5 shorter roots and smaller offshoots were observed at 31.3 mg/l. At 100 mg/l fronds were curved and coloured red with smaller offshoots.
The roots were shortened. On day 7 partly shorter roots were observed at 9.77 mg/l. At 31.3 mg/l the fronds were partly coloured red with smaller offshoots.
The roots were shortened. At 100 mg/l fronds were curved and coloured red with smaller offshoots. The roots were shortened.

MEASURED CONCENTRATIONS
The initial mean measured concentration of test item in the test item solutions was 99 % of nominal. The overall mean concentration of fresh and aged samples was 96 % of nominal.
Toxicological endpoints were evaluated using nominal concentrations of the test item.

Toxicological endpoints

Endpoint Test item [mg/l] nominal
EC10 EC20 EC50  NOEC LOEC1)
Yield of frond numbers 19.91) 32.71) 69.51) 31.32) 1002)
Growth rate of frond numbers 35.81) 55.81) > 100 31.32) 1002)
Yield of dry weight 14.41) 23.81) 50.61) 9.773) 31.33)
Growth rate of dry weight 26.71) 41.31) 79.81) 9.773) 31.33)

1) Probit analysis following the Gompertz distribution

2) following Dunnetts-t-test

3) following Jonckheere-Terpstra

Frond numbers: mean values

Nominal conc. mg/l Mean of frond numbers Yield based on mean frond numbers Inhibition [%]
0 d 3 d 5 d 7 d 7-0 d
Control 12 44 82 138 126 0.0
0.954 12 42 79 133 121 4.0
3.05 12 42 84 143 131 -4.0
9.77 12 40 78 133 121 4.0
31.3 12 39 75 131 119 5.6
100 12 27 35 42 30* 76.2

* significant differences compared to control (following Dunnett´s t-test)

Frond numbers: mean growth rates

Nominal conc. mg/l Mean growth rate μ [d-1] % Inhibition Growth rate [μ]
3 d 5 d 7 d 3 d 5 d 7 d
Control 0.4289 0.3845 0.3488 0.0 0.0 0.0
0.954 0.4198 0.3768 0.3435 2.1 2.0 1.5
3.05 0.4175 0.3890 0.3539 2.7 -1.2 -1.5
9.77 0.3976 0.3739 0.3436 7.3 2.8 1.5
31.3 0.3924 0.3655 0.3413 8.5 4.9 2.2
100 0.2661 0.2120 0.1800* 38.0 44.9 48.4

* significant differences compared to control (following Dunnetts-t-test)

Frond numbers: mean doubling times (Td)

Nominal conc. mg/l Td [days]
3 d 5 d 7 d 7 h
Control 1.616 1.803 1.987 47.7
0.954 1.651 1.840 2.018 48.4
3.05 1.660 1.782 1.959 47.0
9.77 1.743 1.854 2.017 48.4
31.3 1.766 1.896 2.031 48.7
100 2.605 3.270 3.851 92.4

Dry weight: mean values and yield

Nominal conc. mg/l Mean dry weight [g] Yield based on dry weight: inhibition %
0 d 7 d 7 - 0 d 7 - 0 d
Control 0.0031 0.0326 0.295 0
0.954 0.0337 0.0306 -3.7
3.05 0.0346 0.0315 -6.8
9.77 0.0297 0.0266 9.8
31.3 0.0255 0.0224* 24.1
100 0.0071 0.004 86.4

* significant differences compared to control (following Jonckheere-Terpstra)

Dry weight: mean growth rate and inhibition of growth rate

Nominal conc. mg/l Mean growth rate μ [d-1] Inhibition growth rate [%]
Day 7
Control 0.3349 0.0
0.954 0.3408 -1.8
3.05 0.3444 -2.8
9.77 0.3226 3.7
31.3 0.3010* 10.1
100 0.1177* 64.9

* significant differences compared to control (following Jonckheere-Terpstra)

MEASURED CONCENTRATIONS

Test item nominal [mg/l] Test item found
Sampling mg a.i./l % of nominal Geometric mean %
Control 0 d fresh n.d. - -
3 d aged n.d. -
3 d fresh n.d. -
5 d aged n.d. -
5 d fresh n.d. -
0.954 0 d fresh 0.928 97 91
3 d aged 0.824 86
3 d fresh 0.905 95
5 d aged 0.809 85
5 d fresh 0.953 100
3.05 0 d fresh 2.82 92 91
3 d aged 2.90 95
3 d fresh 2.86 94
5 d aged 2.66 87
5 d fresh 2.72 89
9.77 0 d fresh 9.51 97 96
3 d aged 9.40 96
3 d fresh 9.90 101
5 d aged 8.72 89
5 d fresh 9.98 102
31.3 0 d fresh 32.1 103 99
3 d aged 30.5 97
3 d fresh 31.7 101
5 d aged 29.2 93
5 d fresh 32.9 105
100 0 d fresh 105 105 100
3 d aged 99.6 100
3 d fresh 100 100
5 d aged 98.8 99
5 d fresh 101 101

- = not calculated; n.d. = not detectable; LOQ = 0.800 mg/l test item; LOD = 0.240 mg/l test item

Validity criteria fulfilled:
yes
Remarks:
doubling time of control frond numbers was calculated to be 47.7 h, corresponding to 1.987 days at day 7
Conclusions:
EC50 (7d) > 100 mg/l (nominal), based on growth rate of frond number
EC50 (7d): 79.8 mg/l (nominal), based on growth rate of dry weight
Executive summary:

The effect of the test item was evaluated in a growth inhibition test using the aquatic plant Lemna gibba (duckweed). The test was performed according to OECD guideline 221, 2006. A semi-static test with nominal concentrations of 0 (control), 0.954, 3.05, 9.77, 31.3 and 100 mg/l was performed. Vegetative growth was observed for a test period of 7 days by measuring the increase of frond numbers and dry weight. The following endpoints (NOEC/LOEC, EC10, 20, 50) were determined: frond numbers and dry weight; growth rates of frond numbers and dry weight; yield of frond numbers and dry weight; doubling times of frond numbers.

The initial mean measured concentration of test item in the test item solutions was 99 % of nominal. The overall mean concentration of fresh and aged samples was 96 % of nominal. Toxicological endpoints were evaluated using nominal concentrations of test item.

Significant inhibitory effects were determined for yield of frond numbers and for growth rate of frond numbers at 100 mg/l (nominal). On day 3 slightly shorter roots were observed at 31.3 mg/l. At 100 mg/l the fronds were curved and coloured red and offshoots were smaller. The roots were shortened. On day 5 shorter roots and smaller offshoots were observed at 31.3 mg/l. At 100 mg/l fronds were curved and coloured red with smaller offshoots. The roots were shortened. On day 7 partly shorter roots were observed at 9.77 mg/l. At 31.3 mg/l the fronds were partly coloured red with smaller offshoots. The roots were shortened. At 100 mg/l fronds were curved and coloured red with smaller offshoots. The roots were shortened.

The toxicological endpoints were reported to be

Endpoint Test item [mg/l] nominal
EC10 EC20 EC50  NOEC LOEC1)
Yield of frond numbers 19.91) 32.71) 69.51) 31.32) 1002)
Growth rate of frond numbers 35.81) 55.81) > 100 31.32) 1002)
Yield of dry weight 14.41) 23.81) 50.61) 9.773) 31.33)
Growth rate of dry weight 26.71) 41.31) 79.81) 9.773) 31.33)

1) Probit analysis following the Gompertz distribution

2) following Dunnetts-t-test

3) following Jonckheere-Terpstra

Conclusion

EC50 (7d) > 100 mg/l (nominal), based on growth rate of frond number

EC50 (7d): 79.8 mg/l (nominal), based on growth rate of dry weight

Description of key information

EC50 (7d) > 100 mg/l (nominal), based on growth rate of frond number

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:
31.3 mg/L

Additional information

There are no information about the toxicity potential to aquatic plants of Acid Red 057, thus the available data on structural analogous Similar Substance 01 has been taken into account. The read across approach can be considered as reliable and suitable for the purpose; details and explanations are included in the report attached to the IUCLID section 13.

Potential effects of Similar Substance 01 were evaluated in a growth inhibition test using the aquatic plant Lemna gibba (duckweed). A semi-static test with nominal concentrations of 0 (control), 0.954, 3.05, 9.77, 31.3 and 100 mg/l was performed.

The initial mean measured concentration of test item in the test item solutions was 99 % of nominal. The overall mean concentration of fresh and aged samples was 96 % of nominal. Toxicological endpoints were evaluated using nominal concentrations of test item.

On day 3 slightly shorter roots were observed at 31.3 mg/l. At 100 mg/l the fronds were curved and coloured red and offshoots were smaller. The roots were shortened. On day 5 shorter roots and smaller offshoots were observed at 31.3 mg/l. At 100 mg/l fronds were curved and coloured red with smaller offshoots. The roots were shortened. On day 7 partly shorter roots were observed at 9.77 mg/l. At 31.3 mg/l the fronds were partly coloured red with smaller offshoots. The roots were shortened. At 100 mg/l fronds were curved and coloured red with smaller offshoots. The roots were shortened.

Significant inhibitory effects were determined for yield of frond numbers and for growth rate of frond numbers at 100 mg/l (nominal): after 7 days of exposure, an inhinibition of 76.2 % was recorded, based on the frond number yield. At the same concentration, the inhibition of the growth rate estimated on the basis of the frond number was 48.4 %. At the nominal concentration of 100 mg/l, data on yield and growth rate of dry weight indicated an inhibition of 86.4 % and 64.9 %, respectively.