Iodomethane

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:

experimental study

Adequacy of study:

key study

Study period:

17 February 2006 to 02 July 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.4400 (Aquatic Plant Toxicity Test using Lemna spp. Tiers I & II))

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Samples of new test solutions were collected at test initiation and on Days 3 and 5 while samples of old solutions were collected on Days 3 and 5 and at test termination to measure concentrations of the test material. Samples of new test solutions were collected from the test solution of the surrogate bottle prepared for each treatment and control group. Samples of old solutions were collected from an indiscriminate replicate of each treatment and control group.
- All samples were collected from the closed bottle through a Teflon lined septa and analysed immediately without storage.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- A primary stock solution was prepared at a nominal concentration of 268 mg a.i./L by accurately measuring 29.6 µL of the test material and adding it to 20X AAP medium. The amount of test material measured was based on the specific gravity (2.27) of the test material. The primary stock solution was sonicated approximately 5 minutes and inverted at least 20 times and appeared clear and colourless. Each respective test replicate was prepared individually by diluting aliquots of the primary stock solution to a total volume of 200 mL with 20X AAP algal medium. Test solutions were prepared at nominal concentrations of 0.18, 0.41, 0.91, 2.0, 4.5 and 10 mg a.i./L. All test solutions appeared clear and colourless. New test solutions were prepared on Days 0, 3 and 5 of the study.

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: Duck weed (Lemna gibba)
- Strain: G3
- Source: Original duckweed cultures were from the US Department of Agriculture and have been maintained in culture by Wildlife International Ltd
- Age of inoculum: Duckweed plants used in the test were from cultures that had been actively growing in Lemna (20X AAP) culture medium for at least 2 weeks prior to test initiation.
- Method of cultivation: Duck weed plants were cultured and tested in 20X AAP medium. Stock nutrient solutions were added to purified well water and the pH was adjusted to 7.5 using 10 % HCl.

Test type:

static

Water media type:

freshwater

Total exposure duration:

7 d

Test temperature:

25 ± 2 °C

pH:

7.5-7.9 in new solutions and 8.2-8.8 in old test solutions

Nominal and measured concentrations:

- Nominal concentration: 0.18, 0.41, 0.91, 2.0, 4.5 and 10.0 mg/L
- Mean measured test concentations: 0.17, 0.38, 0.85, 1.7, 3.9 and 9.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile 300 mL glass bottles with screw cap with Teflon lined septum containing 200 mL of test solution or control medium.
- Renewal rate of test solution: New solution was added on days 3 and 5
- No. of fronds per colony: Colonies began with 12 fronds (4 plants) in each culture

GROWTH MEDIUM
- AAP medium diluted in Wildlife International Ltd., purified well water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: culture medium pH adjusted to 7.5
- Photoperiod: Continuous light
- Light intensity and quality: 5000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Growth, defined as an increase in the total number of fronds in each replicate test chamber, was determined through direct counts on Days 0, 3 and 5 during the test and at the end of the test for all treatment and control replicates. In addition, the total number of duckweed plants in each replicate test chamber was determined at test termination. Observations of effects such as chlorosis, necrosis, dead fronds, root destruction and break-up of duckweed colonies were performed on Days 0, 3 and 5 during the test and at the end of the test. Chlorotic fronds were defined as fronds possessing areas of bleached colour progressing from green to yellow. Necrotic fronds were those with localized regions of dead or decaying tissue, usually surrounded by healthy tissue. Fronds with no apparent living tissue, usually all brown, black, white, yellow or clear, were considered to be dead. Any other abnormalities in frond or plant appearance were also documented.
- Biomass (dry weight in milligrams) was determined at the beginning of the test from three samples (12 fronds/4 plants) of the inoculum culture, and at the end of the exposure period with the plant material from each test and control chamber after completion of the final-day frond count. The plants used in the determination . of biomass were transferred to pre-weighed aluminium pans and dried at approximately 60 °C for approximately 48 hours prior to the final weighing.

Reference substance (positive control):

no

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL 2.3-2.9 mg/L

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.38 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

MEASUREMENT OF TEST CONCENTRATIONS
- Nominal concentrations of the measured test solutions sampled were 0.18, 0.41, 0.91, 2.0, 4.5 and 10 mg a.i./L. The analytical results of the new solutions ranged from 73 to 108 % of nominal. The analytical results of the old solutions ranged from 75 to 108 % of nominal, respectively. Mean measured test concentrations of the treatment levels were 0.17, 0.38, 0.85, 1.7, 3.9 and 9 .0 mg a.i./L representing 94, 93, 93, 85, 84 and 90 % of nominal, respectively. EC, NOAEC and WEC values are based on mean measured concentrations of the solutions.

OBSERVATIONS AND MEASUREMENTS
- Temperatures ranged from 25.2 to 25.6 °C and were within the range established for the test. Measurements of test solution pH ranged from 7.5 to 7.9 in the new test solutions on Days 0, 3 and 5 and ranged from 8.2 to 8.8 in the old test solutions on Days 3, 5 and at test termination. Increases in pH were observed in the test solutions over the duration of the study because photosynthetic activity of aquatic plants increases pH. Light intensity ranged from 4520 to 5520 lux and was within the range established for the test (5000 ± 750 lux).
- The toxicity of the test material to duckweed was determined by evaluating the production of plants and fronds and their general health over a 7-day exposure period.
- Duckweed plants in each control replicate exhibited normal growth throughout the test. The doubling time of frond number in the control was 2.3 days, which met the validity criteria for the test of <2.5 days. After 7 days of exposure, there were no noticeable changes in frond or plant appearance in the ≤ 1.7 mg a.i./L treatment group when compared to the control. At concentrations of 3.9 mg a.i./L and above, treatment-related effects on fronds and plants were evident, including small fronds, root destruction and/or breakup of colonies in comparison to the control. A noticeable increase in chlorotic fronds and dead fronds were observed in the 3.9 and 9.0 mg a.i./L treatment levels.
- Percent inhibition of frond number in the 0.17, 0.38, 0.85, 1.7, 3.9 and 9.0 mg a.i./L treatment groups at test termination was 0. 67, 3. 7, 13, 38, 67 and 88 %, respectively, when compared to the control. Percent inhibition of growth rate based on frond number in the 0.17, 0.38, 0.85, 1.7, 3.9 and 9.0 mg a.i./L treatment groups at test termination was 0.33, 1.8, 6.5, 22, 52 and 99 % respectively, when compared to the control. Treatment related effects were apparent in the four highest test concentrations. Dunnett's t­test indicated that frond number and growth rate based on frond number were significantly reduced (p<0.05) in the 0.85, 1.7, 3.9 and 9.0 mg a.i./L treatment levels. Consequently the Day 7 NOAEC and LOEC based on statistical evaluation for frond number and growth rate based on frond number was 0.38 and 0.85 mg a.i./L, respectively.
-Percent inhibition of biomass in the 0.17, 0.38, 0.85, 1.7, 3.9 and 9.0 mg a.i./L treatment groups at test termination was -2.6, -1.1, 8.1, 20, 49 and 100 %, respectively, when compared to the control. Treatment related effects were apparent in the four highest test concentrations for biomass. Dunnett's t­test indicated that biomass was significantly reduced (p<0.05) in the 0.85, 1.7, 3.9 and 9.0 mg a.i./L treatment levels. Consequently the Day 7 NOAEC and LOEC for biomass were 0.38 and 0.85 mg a.i./L, respectively. Percent inhibition of growth rate based on biomass in the 0.17, 0.38, 0.85, 1.7, 3.9 and 9.0 mg a.i./L treatment groups at test termination was -1.1, -0.49, 3.7, 9.7, 28 and 100 %, respectively, when compared to the control. Treatment related effects were apparent in the three highest test concentrations for biomass. Dunnett's t-test indicated that growth rate based on biomass was significantly reduced (p<0.05) in the 1.7, 3.9 and 9.0 mg a.i./L treatment levels. Consequently the Day 7 NOAEC and LOEC for growth rate based on biomass were 0.85 and 1. 7 mg a.i./L, respectively.

Reported statistics and error estimates:

The percentages of dead, chlorotic and necrotic fronds were calculated as the number of dead, chlorotic and necrotic fronds relative to the total number of fronds present in each test chamber. The Day 7 frond numbers, growth rates and biomass were evaluated for normality and homogeneity of variances (p = 0.01) using the Shapiro-Wilk' s and Bartlett's tests, respectively. Treatment groups were compared to the control groups (p=0.05) using analysis of variance (ANOVA) and Dunnett's t-test. The minimum significant difference (MSD) detected by ANOVA for fronds number was 6.5, for growth rate based on frond number was 0.015, for biomass was 0.62 and for growth rate based on biomass was 0.011. Day 7 EC50 values were determined using linear interpolation with treatment response (frond number, biomass and growth rates) and exposure concentration data. Results of the statistical analyses, as well as an evaluation of the concentration-response pattern and other observations of effects, were used in the determination of the NOAEC and LOEC.

Table 2: Mean frond numbers, biomass and percent inhibition values

Mean measured test concentration (mg a.s./L)	Day 3 frond number		Day 5 frond number		Day 7 frond number		Final biomass
	Mean Frond no.	% inhibition	Mean Frond no.	% inhibition	Mean Frond no.	% inhibition	Mean biomass (mg)	% inhibition
0 (Control)	30	-	58	-	99	-	9.0	-
0.17	29	1.1	56	2.3	98	0.67	9.3	-2.6
0.38	28	5.6	57	1.2	95	3.7	9.1	-1.1
0.85	26	13	51	12	86*	13	8.3*	8.1
1.7	24	18	41	29	62*	38	7.2*	20
3.9	19	37	26	55	33*	67	4.6*	49
9.0	13	56	14	76	12*	88	0*	100
	EC50= 2.6 mg/L (95% CI = 2.3 to 2.9 mg/L)						EC50= 3.9 mg/L (95% CI = 3.2 to 4.7 mg/L)

* Statistically significant difference on Day 7 frond growth and biomass (p<0.05) from the Control using Dunnett’s t-test.

Table 3: Mean frond numbers and biomass growth rates and percent inhibition values

Mean measured test concentration (mg a.s./L)	Day 3 frond number		Day 5 frond number		Day 7 frond number		Final biomass
	Mean Frond no.	% inhibition	Mean Frond no.	% inhibition	Mean Frond no.	% inhibition	Mean biomass (mg)	% inhibition
0 (Control)	0.301	-	0.314	-	0.301	-	0.276	-
0.17	0.298	1.2	0.309	1.5	0.300	0.33	0.279	-1.1
0.38	0.282	6.4	0.311	0.75	0.296	1.8	0.277	-0.49
0.85	0.253	16	0.289	7.9	0.282*	6.5	0.265	3.7
1.7	0.236	22	0.244	22	0.234*	22	0.249*	9.7
3.9	0.144	52	0.154	51	0.144*	52	0.198*	28
9.0	0.025	92	0.025	92	0.003*	99	0.00*	100
	EC50= 3.7 mg/L (95% CI = 3.5 to 4.1 mg/L)						EC50= 5.4 mg/L(95% CI = 5.1 to 5.8 mg/L)

* Statistically significant difference on Day 7 frond growth rate and biomass (p<0.05) from the Control using Dunnett’s t-test.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this 7-day static-renewal study with the aquatic macrophyte (Lemna gibba) the 7-day EC50 value was 2.6 mg/L. This was based on frond number and represented the worst case endpoint. The corresponding NOEC was considered to be equivalent to 0.38 mg/L.

Executive summary:

The toxicity of the test material to aquatic plants was determined in accordance with the standardised guidelines OECD 221 and EPA OPPTS 850.4400, under GLP conditions.

Fronds of duckweed (Lemna gibbaG3) were exposed to a geometric series of six test concentrations, and a negative control (culture medium) under static renewal conditions for 7 days. The nominal test concentrations were 0.18, 0.41, 0.91, 2.0, 4.5 and 10 mg/L. At the start of exposure (Day 0) four duckweed plants were added to each replicate container. Duckweed plants were transferred to newly prepared test solutions on Days 3 and 5 of the test. 

Measured test concentrations were determined from samples of test medium collected from each treatment and control group at the beginning of the test, on Days 3 and 5 “new” and “old” test solutions and at test termination. The mean measured concentrations of the test material were: 0.17, 0.38, 0.85, 1.7, 3.9 and 9.0 mg/L at nominal concentrations of 0.18, 0.41, 0.91, 2.0, 4.5 and 10 mg/L, respectively.

Growth, defined as an increase in the total number of fronds in each replicate test chamber, was determined through direct counts on Days 0, 3 and 5 during the test and at the end of the test. In addition, the total number of duckweed plants in each replicate test chamber was determined at the end of the test. Observations of effects such as chlorosis, necrosis, dead fronds, root destruction and break-up of duckweed colonies were performed on Days 0, 3, 5 and at the end of the test. Biomass was determined at the beginning and end of the test.

The 7‑day EC₅₀value was 2.6 mg /L, based on frond number. For the test to be valid, the doubling time of frond number in the control must be less than 2.5 days (60 h), corresponding to approximately a seven-fold increase in seven days. This was achieved and the test was therefore considered valid.

Under the conditions of this 7-day static-renewal study with the aquatic macrophyte (Lemna gibba) the 7-day EC50 value was 2.6 mg/L. This was based on frond number and represented the worst case endpoint. The corresponding NOEC was considered to be equivalent to 0.38 mg/L.

Description of key information

Under the conditions of this 7-day static-renewal study with the aquatic macrophyte (Lemna gibba) the 7-day EC50 value was 2.6 mg/L. This was based on frond number and represented the worst case endpoint. The corresponding NOEC was considered to be equivalent to 0.38 mg/L.

Key value for chemical safety assessment

EC50 for freshwater plants:

2.6 mg/L

EC10 or NOEC for freshwater plants:

0.38 mg/L

Additional information

The toxicity of the test material to aquatic plants was determined in accordance with the standardised guidelines OECD 221 and EPA OPPTS 850.4400, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Fronds of duckweed (Lemna gibbaG3) were exposed to a geometric series of six test concentrations, and a negative control (culture medium) under static renewal conditions for 7 days. The nominal test concentrations were 0.18, 0.41, 0.91, 2.0, 4.5 and 10 mg/L. At the start of exposure (Day 0) four duckweed plants were added to each replicate container. Duckweed plants were transferred to newly prepared test solutions on Days 3 and 5 of the test. 

Measured test concentrations were determined from samples of test medium collected from each treatment and control group at the beginning of the test, on Days 3 and 5 “new” and “old” test solutions and at test termination. The mean measured concentrations of the test material were: 0.17, 0.38, 0.85, 1.7, 3.9 and 9.0 mg/L at nominal concentrations of 0.18, 0.41, 0.91, 2.0, 4.5 and 10 mg/L, respectively.

Growth, defined as an increase in the total number of fronds in each replicate test chamber, was determined through direct counts on Days 0, 3 and 5 during the test and at the end of the test. In addition, the total number of duckweed plants in each replicate test chamber was determined at the end of the test. Observations of effects such as chlorosis, necrosis, dead fronds, root destruction and break-up of duckweed colonies were performed on Days 0, 3, 5 and at the end of the test. Biomass was determined at the beginning and end of the test.

The 7‑day EC₅₀value was 2.6 mg /L, based on frond number. For the test to be valid, the doubling time of frond number in the control must be less than 2.5 days (60 h), corresponding to approximately a seven-fold increase in seven days. This was achieved and the test was therefore considered valid.

Under the conditions of this 7-day static-renewal study with the aquatic macrophyte (Lemna gibba) the 7-day EC50 value was 2.6 mg/L. This was based on frond number and represented the worst case endpoint. The corresponding NOEC was considered to be equivalent to 0.38 mg/L.