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Toxicity to aquatic plants other than algae

Link to relevant study record(s)

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 September 23, 2015 to October 08, 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was performed on a similar substance. The complete justification for the read across approach is attached at section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

100.03 mg of the test item was weighed and made up to 100 ml using 20X-AAP growth medium to make a stock concentration of 1 mg/ml.
All the required test concentrations were prepared for 800 ml. From the stock solution, each required test concentration was prepared as follows: 800 µl, 1760 µl, 3872 µl, 8520 µl, 18744 µl and 41232 µl were taken and made up to 800 ml with 20X-AAP medium to obtain 1, 2.2, 4.84, 10.65, 23.43 and 51.54 mg/l concentrations respectively.
For Control, 20X-AAP medium was used.

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Strain: Lemna gibba (Duckweed)
- Source (laboratory, culture collection): In-house culture, RCC Laboratories India Private Limited (Originally procured by Department of Plant Biology & Plant Biotechnology Madras Christian College, Chennai)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Post exposure observation period:

Frond number in each replicate was counted on days ‘0’, ‘3’, ‘5’ and ‘7’. A representative sample (approximately 12 fronds) of pre-culture was checked for its average dry weight on day ‘0’ of exposure and dry weight of plant material from each test and control vessel on day ‘7’ of exposure. Observation was performed on day ‘0’, ‘3’, ‘5’ and ‘7’ for the abnormal appearance or any developmental changes due to the effect of the test item (if any).

Hardness:

None

Test temperature:

Temperature in test vessel (°C)
Start of the test Control : 23.5
Treatment: 23.5
End of the test
Control: 24.8-25.1
Treatment: 25.1-25.3

pH:

Medium pH range Start of the test
Control : 7.54
Treatment: 7.48
End of the test
Control: 8.69-8.72
Treatment: 8.72-8.75

Nominal and measured concentrations:

Based on the results of the range finding experiment, six test concentrations were fixed in a geometric series with a spacing factor of 2.2 for the main experiment.

Details on test conditions:

PRE-CULTURE
Prior to start of the test, sufficient colonies were transferred into fresh sterile medium and the preculture was maintained for 8 days under the test conditions.

TEST SYSTEM
- Test vessel: 500 ml Glass Beaker
- No. of organisms per vessel: 12 fronds (3 plants)
- No. of vessels per concentration: (Main Experiment) 3 replicates for each concentration
- No. of vessels per control: 6 Replicates.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: At the beginning of the test
- Photoperiod: Continuous Light
- Light intensity and quality: 8460-8650 Lux

TEST CONCENTRATIONS
- For range finding experiment the concentrations selected were 0.1, 1, 10, 25, 50 and 100 mg/l.
- For Limit concentration ,100 mg/l which is equivalent to 130mg/1300ml. 130.04 mg of the test item was dissolved and made up to 1300 ml of 20X-AAP medium. From this 1200 ml of stock was directly used. For Control, 20X-AAP medium was used.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 22.14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: confidence limit 22.14 - 20.14

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 9.55 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: confidence limit 9.92 - 9.19

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 21.67 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Frond Dry Weight; Growth

Remarks on result:

other: confidence limit 22.80 - 20.59

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 8.69 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Frond Dry Weight; Yield

Remarks on result:

other: confidence limit 9.03 - 8.35

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

ca. 2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Results with reference substance (positive control):

ErC50 of 3,5-Dichlorophenol based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 4.30 mg/l with a lower confidence limit of 4.15 mg/L and the upper confidence limit of 4.46 mg/L. EyC50 of 3,5-Dichlorophenol based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 2.38 mg/l with a lower confidence limit of 2.29 mg/l and the upper confidence limit of 2.48 mg/l. ErC50 of 3,5-Dichlorophenol based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was calculated as 3.02 mg/l with a lower confidence limit of 2.89 mg/l and the upper confidence limit of 3.16 mg/l. EyC50 of 3,5-Dichlorophenol based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was calculated as 1.53 mg/l with a lower confidence limit of 1.46 mg/l and the upper confidence limit of 1.59 mg/l.

Reported statistics and error estimates:

Mean coefficient of variation of average specific growth rate in replicate control and treated cultures were calculated. No analysis was done as the study is concluded with the Limit Test.
NOMINAL: 1, 2.2, 4.84, 10.65, 23.43 and 51.54 mg/l

BASED ON FROND NUMBERS:

The percent inhibition on the average specific growth rate on day ‘7’ for the test concentrations of 1, 2.2, 4.3.4, 10.65, 23.43 and 51.54 mg/l was calculated as 0.12%, 0.11%, 8.33%, 34.62%, 46.54% and 80.82% respectively.

BASED ON FROND DRY WEIGHT:

The percent inhibition on the average growth rate on day ‘7’ for the concentration of 1, 2.2, 4.84, 10.65, 23.43 and 51.54 mg/L was calculated as 0%, 0.43%, 11.75%, 35.04%, 42.09% and 80.13% respectively.

Validity criteria fulfilled:

yes

Conclusions:

ErC50(frond numbers) 21.12 mg/l
ErC50(frond dry weight) 21.67 mg/l
EyC50(frond numbers) 9.55 mg/l
EyC50(frond dry weight) 8.69 mg/l

Executive summary:

Lemna gibba (Duckweed) were exposed to the test item at test concentration 100 mg/l. Initially on day ‘0’, 12 fronds were inoculated in beaker containing 200 ml of test medium (20X-AAP medium). Three replicatesfor the main experiment and six replicates for control were maintained. The test item was formulated in 20X-AAP medium.

Prior to exposure, Lemna gibba was pre-cultured in the 20X-AAP medium for period of ‘8’ days and the pre-culture was found healthy.

After exposure, Lemna gibba was observed for frond count on days ‘3’, ‘5’ and ‘7’. Dry weight of the fronds were recorded at start (pre-culture representative samples) and at the end of the test.

Fronds were observed for their appearance on days ‘3’, ‘5’ and ‘7’. All fronds were found normal and healthy in all replicates of control and test concentrations except 51.54 mg/L where necrosis observed on day ‘5’ and in addition to necrosis, root filaments were turned blue in 10.65, 23.43 and 51.54 mg/l concentrations on day ‘7’.

The doubling time (Td) of frond number in the control group was calculated as 1.608 day^-1.

Frond Numbers:

Growht rate

From the average specific growth rate recorded with the test concentration 100 mg/l the ErC50 was calculated as 21.12 mg/l.

Yield

From the yield recorded with the test concentration 100 mg/l the ErC50 was calculated as 9.55 mg/l.

Frond Dry Weight:

Growht rate

From the average specific growth rate recorded with the test concentration 100 mg/l the ErC50 was calculated as 21.67 mg/l.

Yield

From the yield recorded with the test concentration 100 mg/l the ErC50 was calculated as 8.69 mg/l.

Results

ErC50(frond numbers) 21.12 mg/l

ErC50(frond dry weight) 21.67 mg/l

EyC50(frond numbers) 9.55 mg/l

EyC50(frond dry weight) 8.69 mg/l

Description of key information

 ErC50(frond numbers) 21.12 mg/l

Key value for chemical safety assessment

EC50 for freshwater plants:

21.12 mg/L

Additional information

No tests are available on short-term toxicity to invertebrates performed on the substance in itself nevertheless, one test is available on a similar substance. The complete evaluation and justification of the read across approach is attached at section 13.

Method

Lemna gibba (Duckweed) were exposed to the test item at test concentration of 100 mg/l. Three replicates for each test concentration and six replicates for control were maintained. The test item was formulated in 20X-AAP medium. Dry weight of the fronds were recorded at start (pre-culture representative samples) and at the end of the test.

Fronds were observed for their appearance and number on days ‘3’, ‘5’ and ‘7’. All fronds were found normal and healthy in all replicates of control and test concentration.

Observations

Growht rate

From the average specific growth rate recorded with the test concentration 100 mg/l the ErC50 was calculated as 21.12 mg/l for frond number and 21.67 mg/l based on dry weight.

Yield

From the yield recorded with the test concentration 100 mg/l the ErC50 was calculated as 8.69 mg/l for frond number and 8.69 mg/l based on dry weight.

Results

ErC50(frond numbers) 21.12 mg/l

ErC50(frond dry weight) 21.67 mg/l

EyC50(frond numbers) 9.55 mg/l

EyC50(frond dry weight) 8.69 mg/l

The ErC50 (frond number) has been choosed as key value. Based on OECD 221 "The test endpoint is inhibition of growth, expressed as logarithmic increase in measurement variable (average specific growth rate) during the exposure period". The yield is an additional response variable used to fulfil specific regulatory requirements in some countries.

ECx values based upon average specific growth rate (ErCx) will generally be higher than results based upon yield (EyCx) if the test conditions of this Guideline are adhered to, due to the mathematical basis of the respective approaches.

This should not be interpreted as a difference in sensitivity between the two response variables, simply that the values are different mathematically. The concept of average specific growth rate is based on the general exponential growth pattern of duckweed in non-limited cultures, where toxicity is estimated on the basis of the effects on the growth rate, without being dependent on the absolute level of the specific growth rate of the control, slope of th e concentration-response curve or on test duration.

In contrast, results based upon the yield response variable are dependent upon all these other variables. EyCx is dependent on the specific growth rate of the duckweed species used in each test and on the maximum specific growth rate that can vary between species and even different clones. This response variable should not be used for comparing the sensitivity to toxicants among duckweed species or even different clones. While the use of average specific growth rate for estimating toxicity is scientifically preferred, toxicity estimates based on yield are also included in this Guideline to satisfy current regulatory requirements in some countries.

Total frond area:

The total frond area of all colonies may be determined by image analysis. A silhouette of the test vessel and plants can be captured using a video camera (i.e. by placing the vessel on a light box) and the resulting image digitised. By calibration with flat shapes of known area, the total frond area in a test vessel may then be determined.

Dry weight:

All colonies are collected from each of the test vessels and rinsed with distilled or deionised water. They are blotted to remove excess water and then dried at 60 °C to a constant weight. Any root fragments should be included. The dry weight should be expressed to an accuracy of at least 0.1 mg.

The frond number and the frond appearance are judged as more representative than the dry weight, therefore the value of 50 % of inhibition of growth rate based on frond numbers are used as study result.