1-[2-(2-chloroethoxy)phenylsulfonyl]-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea;triasulfuron (ISO)

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:

18 Mar 2002 to 24 Jul 2002

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)

Version / remarks:

Proposal for a new guideline, 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ASTM Guideline E 1415-91

Version / remarks:

1991

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples (2 x approximately 200 mL) were taken from each freshly prepared test solution and stock solution and the control immediately before exposure. Two of the three replicate test vessels were sampled (approximately 200 mL) at the end of exposure.
- Sample storage conditions before analysis: - 18°C to - 22°C

Vehicle:

yes

Remarks:

DMF

Details on test solutions:

PREPARATION OF STOCK AND TEST SOLUTIONS
A first stock solution was prepared by dissolving 1.0 mg test item in 10.0 mL DMF. 0.10 mL of this stock solution was mixed and made up to 10 mL with medium to produce stock solution 2. 2.0 mL of stock solution 2 was mixed and made up to 20 mL with medium to produce stock solution 3. 6.4, 3.2, 1.6, 0.80, 0.40, 0.20 and 0.10 mL of stock solution 3 was mixed and made up to 1000 mL with medium to produce the test solutions. Calculated amounts of vehicle were given into the test solutions to achieve identical vehicle concentrations.

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: Duckweed
- Strain: Lemna Gibba G3
- Source: In-house culture
- Culture: According to the guideline cited; 26 months under test conditions (54 times 5 plants transferred to fresh medium).
- Preculture: 13 days under test conditions

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Hardness:

311 mg CaCO3/L

Test temperature:

23 ± 2 °C

pH:

7.8 - 9.1

Conductivity:

1622 µS/cm

Nominal and measured concentrations:

Nominal concentrations: 0 (blank control), 0 (vehicle control), 0.010, 0.020, 0.040, 0.080, 0.16, 0.32 and 0.64 µg/L.
Measured concentrations day 0: Measured concentrations day 7: See Table 1 in 'Any other information on materials and methods incl. tables'.

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: Yes
- Test vessel: 600 mL glass beakers
- Type: Closed
- Fill volume: 200 mL
- Type of cover: Watch glasses
- Aeration: No
- Agitation: No
- No. of colonies per vessel: 3 to 5
- No. of fronds per colony: 2 to 4
- No. of vessels per concentration: 3
- No. of vessels per control: 3
- No. of vessels per vehicle control: 3

GROWTH MEDIUM
- Standard medium used: Yes, 20 X AAP growth medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted bi-destilled water
- Temperature: Continuously measured in the incubation chamber and in a test vessel with water at test days 0, 1, 2, 3, 4 and 7.
- pH: At days 0, 2, 4 and 7

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination with cold white fluorescent light
- Light intensity at the start: Approximately 6500 Lux

EFFECT PARAMETERS MEASURED
- Determination of frond number: Counted and observed at days 0, 2, 4 and 7
- Determination of biomass: Dry weight at day 7

RANGE FINDING TEST
A range-finding test (test concentrations: 0.1, 0.3, 1.0, 3.0, 10, 30 and 100 μg/L) was conducted to determine a concentration range of the test substance to use in the definitive test. The fronds were counted at the start and at the end of exposure. A growth inhibition was visible at all test concentrations.
Based on the results of the range-finding test, the following nominal concentrations were selected for the definitive test: 0.64, 0.32, 0.16, 0.080, 0.040, 0.020 and 0.010 μg/L.

Reference substance (positive control):

yes

Remarks:

3,5 Dichlorophenole at nominal concentrations of 0.11, 0.33, 1.0, 3.0, 6.0 and 18 mg/L

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

0.28 µg/L

95% CI:

>= 0.24 - <= 0.32

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.08 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

dry weight

frond number

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

0.18 µg/L

95% CI:

>= 0.15 - <= 0.21

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

frond number

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.08 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

frond number

Details on results:

An overview of the results is provided in Table 2 – Table 7 in ‘Any other information on results incl. tables’.
- Number of fronds: Changes in mean number of fronds indicated that exponential growth occurred in the control replicates. A statistically significant (p>0.05) difference in mean frond number (Inhibition, multiple comparison growth rates) between the control and treatments was observed at nominal concentrations of 0.16, 0.32 and 0.64 µg/L. 28.7, 63.7 and 70.6 % inhibition was recorded at these test concentrations.
- Dry weight: The difference between the dry weight of the plants in the controls and the dry weight at the test concentrations was statistically significant at test concentrations of 0.16, 0.32 and 0.64 µg/L.
- Observations: It was visually recorded (after 7 days) that the fronds of the plants were smaller in comparison to controls at nominal concentrations of 0.16, 0.32 and 0.64 µg/L. The plants with treatment groups of 0.16, 0.32 and 0.64 μg/L showed changes in plant development.

Results with reference substance (positive control):

The duckweed Lemna gibba was demonstrated to be adequately sensitive with 3,5 dichlorophenole as reference item.

Reported statistics and error estimates:

The program package ECOS was used to analyse data according to guideline OECD (1996). The effect of the test item on the growth rates and growth areas of Lemna gibba G3 was calculated using logit analysis. For determination of the NOErC/NOEC (AUC) frond numbers and NOEC dry frond weight a Dunnett's Multiple Comparison Test was used. Treatments were compared with the pooled control.

Table 2. Initial frond number at the start of exposure

Concentration nominal µg/L	Fronds/replicate
	1	2	3
Blank	17	17	19
Vehicle	l7	18	19
0.010	20	17	18
0.020	18	18	17
0.040	17	16	19
0.080	17	17	18
0.16	20	18	15
0.32	16	15	15
0.64	19	18	18

Table 3. Frond number after 2 days of exposure

Concentration nominal µg/L	Fronds/repIicate
	1	2	3
Blank	34	38	40
Vehicle	38	38	36
0.010	49	34	37
0.020	40	39	40
0.040	35	32	38
0.080	36	39	40
0.16	41	37	33
0.32	27	28	25
0.64	23	23	26

Table 4. Frond number after 4 days of exposure

 

Concentration nominal µg/L	Fronds/repIicate
	1	2	3
Blank	69	76	90
Vehicle	72	77	84
0.010	101	76	71
0.020	79	77	81
0.040	70	71	82
0.080	76	84	86
0.16	57	54	53
0.32	33	33	35
0.64	31	27	32

Table 5. Frond number after 7 days of exposure

 

Concentration nominal µg/L	Fronds/repIicate
	1	2	3
Blank	223	250	279
Vehicle	263	257	272
0.010	324	272	244
0.020	254	238	254
0.040	258	217	278
0.080	267	271	292
0.16	134	127	118
0.32	43	56	47
0.64	37	40	42

Table 6. Weight of dry fronds after 7 days of exposure

 

Concentration nominal µg/L	Weight of fronds (mg)
	1	2	3	Mean
Blank	31.00	30.80	37.70	33.17
Vehicle	39.50	33.80	35.40	36.23
0.010	42.30	33.30	32.50	36.03
0.020	30.90	31.60	30.60	31.03
0.040	35.70	39.00	38.90	37.87
0.080	35.50	36.70	43.00	38.40
0.16	26.10	25.90	21.30	24.43
0.32	13.30	14.80	14.00	14.03
0.64	12.40	13.30	13.10	12.93

Table 7. Effect parameters

 

Effect parameter	Inhibition	Inhibition
	(growth rates)	(AUC)
EC 50 (0 - 7 d) number of fronds:	0.28 μg/L	0.18 μg/L
95 % confidence limit :	0.24 - 0.32 μg/L	0.15 - 0.21 μg/L
slope:	1.733 ± 0.199	2.946 ± 0.631
NOEC (0 - 7 d) number of fronds:	0.080 μg/L	0.080 μg/L
NOEC (7d), dry frond weight:	0.080 μg/L
(significant on 5 % level)

Validity criteria

The doubling times of the frond numbers in the control was less than 2 days (corresponding to approximately an 8-fold increase in seven days) corresponding to an approximately 14.2-fold increase in biomass in 7 days with the test item, therefore the test is valid.

Validity criteria fulfilled:

yes

Remarks:

See section 'Any other information on results incl. tables'.

Conclusions:

In a growth inhibition study with aquatic plants following OECD TG 221 (draft), OPPTS 850.4400 and ASTM Guideline E 1415-91, the 7-day ErC50 and NOErC of frond numbers of Lemna gibba were 0.28 and 0.080 µg/L, respectively, based on nominal concentrations.

Executive summary:

This study was conducted to determine the toxicity of the test item on growth of the freshwater plant Lemna gibba according to OECD TG 221 (draft), OPPTS 850.4400 and ASTM Guideline E 1415-91, and in compliance with GLP principles. Lemna plants were exposed for 7 days in a static system at nominal test concentrations of 0 (blank control), 0 (vehicle control), 0.010, 0.020, 0.040, 0.080, 0.16, 0.32 and 0.64 µg/L with an initial frond number of 15-20 fronds per replicate. The test concentrations were analysed at 0 and 7 days by using LC/LC-ESI/MS/MS-detection. The measured concentrations were between 98 and 145% of the nominal values at the start and between 105 and 130% at the end of the test. The test was carried out under the following conditions: temperature 23 ± 2 °C, continuous light (approximately 6500 Lux), pH 7.8 - 7.9 at the start and pH 8.8 – 9.1 at the end of the test. 

After 7 days of exposure, the mean frond number was 250.7, 264.0, 280.0, 248.7, 251.0, 276.7, 126.3, 48.7 and 39.7 in blank control, vehicle control, 0.010, 0.020, 0.040, 0.080, 0.16, 0.32 and 0.64 µg/L treatments, respectively. When compared to mean frond number in the pooled control, the 0.010, 0.020, 0.040, 0.080, 0.16, 0.32 and 0.64 µg/L treatments showed a growth rate inhibition of 0, 1.83, 0.56, 0, 28.69, 63.69 and 70.63%, respectively. After 7 days of exposure the mean biomass, given as AUC, was 33.17, 36.23, 36.03, 31.03, 37.87, 38.40, 24.43, 14.03 and 12.93 mg in blank control, vehicle control, 0.010, 0.020, 0.040, 0.080, 0.16, 0.32 and 0.64 µg/L treatments, respectively. When compared to the biomass in the pooled control, the 0.010, 0.020, 0.040, 0.080, 0.16, 0.32 and 0.64 µg/L treatments showed an inhibition of biomass of 0, 1.07, 4.26, 0, 46.65, 81.28 and 86.40%, respectively. Based on nominal concentrations, the 7-day ErC50 was 0.28 µg/L, the EbC50 was 0.18 µg/L and the NOEC for growth rate inhibition of frond numbers, dry frond weight and inhibition of biomass was 0.080 µg/L.

Description of key information

All available data was assessed. Different plant species and different exposure periods were used. Thus, the study following a standard guideline and with the highest reliability was included here as the key study. Other studies are included as supporting information.

Freshwater, 7-d ErC50 0.28 µg/L, 7-d NOErC 0.080 µg/L, static, Lemna gibba, growth rate of frond numbers, OECD TG 221 (draft), Grade 2002.

Key value for chemical safety assessment

EC50 for freshwater plants:

0.28 µg/L

EC10 or NOEC for freshwater plants:

0.08 µg/L

Additional information

There are four studies available for this endpoint. All of them were performed under GLP but only two of them were performed according to standard guidelines. The 7-d study (Grade 2002) on Lemna gibba was selected as the key study because it followed a standard guideline and it has the highest reliability. Lemna were exposed in a static test to the test item at nominal concentrations of 0 (blank control), 0 (vehicle control), 0.010, 0.020, 0.040, 0.080, 0.16, 0.32 and 0.64 µg/L. Measured concentrations day 0: <LOQ, <LOQ, <LOQ, 0.029, 0.043, 0.078, 0.19, 0.32 and 0.65 µg/L. Measured concentrations day 7: <LOQ, 0.028, <LOQ, 0.026, 0.049, 0.10, 0.20, 0.34 and 0.67 µg/L The initial frond number per replicate was 15 - 20. Three replicates were introduced per treatment and control. The test conditions were as follows: temperature 23 ± 2 °C, continuous light (approximately 6500 Lux), pH 7.8 - 7.9 at the start and pH 8.8 – 9.1 at the end of the test. Fronds were counted and observed at days 0, 2, 4 and 7. Dry weight was determined at day 7. Based on nominal concentrations, the 7-day ErC50 was 0.28 µg/L, the EbC50 was 0.18 µg/L and the NOEC for growth rate inhibition of frond numbers, dry frond weight and inhibition of biomass was 0.080 µg/L.

The other three studies are included as supporting studies. Lemna gibba was exposed for 14 days in a semi-static system. Nominal concentrations were 0.0056,  0.010, 0.018, 0.032, 0.056, 0.10 and 0.18 µg/L. The nominal test concentrations were below the limit of detection (54 µg/L), therefore, samples of nominal 900000, 1800 and 180 µg/L stock solutions were measured and showed that the actual concentrations were between 99 to 111 % of the nominal values. The 14-d EyC50 values for frond number and dry weight were 0.073 and 0.068 µg/L, respectively. The NOEyC value for both frond number and dry weight was 0.032 µg/L (Croudace 1995). Elodea canadensis was exposed for 25 days in a static system. Nominal concentrations were 0.01, 0.03, 0.09, 0.27 and 0.81 µg/L. The mean measured concentrations were 0.0077, 0.024, 0.077, 0.25 and 0.83 µg test item/L. The 25-d EC50 value for growth (growth rate of total shoot length and biomass) was determined to be > 0.81 µg/L (nominal concentrations). The NOEC value for growth was 0.81 µg/L, based on nominal concentrations (Volz 2003a). Myriophyllum spicatum was exposed for 14 days in a static system. Nominal concentrations were 0.002, 0.006, 0.018, 0.045, 0.16 and 0.49 µg/L. The mean measured concentrations were < LOQ, 0.0042, 0.016, 0.041, 0.14 and 0.41 μg test item/L. The 14-d EC50 value for growth (growth rate of total shoot length and biomass) was determined to be > 0.41 µg/L, based on mean measured concentrations. The NOEC value for growth was 0.41 µg/L, based on mean measured concentrations (Volz 2003b).

Endpoints from the Lemna test performed at pH 7.5 should be used in preference to the test conducted at pH 5 as uptake, and therefore plant toxicity, is expected to decrease as external pH values rise above 5. Weakly acid herbicides such as the sulfonylureas exist in undissociated form under acidic conditions but undergo rapid dissociation at alkaline pH. Triasulfuron is known to ionise at pH values above 5. Given that membranes are more permeable to uncharged molecules, initial sulfonylurea uptake by passive diffusion decreases with increasing external pH. Continued uptake against a concentration gradient is possible by the process of ion-trapping, which requires ATP.

5 aquatic toxicity studies on Lemna gibba for individual degradation products (one study for each metabolite) were available. The toxicity to the aquatic plant Lemna gibba was determined in a 7-day static test or semi-static test. For frond number, the 7-day EC50 for biomass (EbC50) and growth rate (ErC50) 4.2 to >101 mg/L, showing lower aquatic toxicity of the degradation products than the substance itself.