2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 December 2001 to 31 December 2001

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:

other: EPA OPP 122-2 (Algal Toxicity, Tiers I and II)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 123-2 (Growth and Reproduction of Aquatic Plants (Tier 2))

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

yes

Details on sampling:

At the beginning and end of the testing period (day 0 and day 14), samples were removed from each test solution, control, solvent control and analysed for test material concentrations. Day 0 samples were removed from the newly prepared test solutions prior to division into replicate vessels. Samples were collected at test termination (day 14) from each individual replicate solution taken from each treatment level. The control and solvent control replicate solutions were individually combined and a single composite sample was removed and analysed for these solutions.
Three quality control (QC) samples were prepared at each sampling interval and remained with the appropriate set of exposure solution samples throughout the analytical process. These QC samples were prepared in 20X algal assay procedure (AAP) medium at nominal concentrations similar to the exposure concentration range. Results of the analyses of the QC samples were used to judge the precision and quality control maintained during the analysis of exposure solution samples.

Vehicle:

yes

Details on test solutions:

A 100 mg a.i./L primary stock solution was prepared by placing 0.2118 g (0.2002 as active ingredient) of test material in a 2000-mL volumetric flask and diluting to volume with 20X AAP medium containing 0.10 mL/L of dimethylformamide (DMF). The resulting stock solution was observed to be clear and colourless, with no visible undissolved test material (e.g., precipitate). Test solutions were prepared from dilutions of the 100 mg a.i./L primary stock solution.

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: duckweed
- Strain: strain G3
Stock cultures were grown in 270-mL covered crystallising dishes containing 100 mL of medium. The cultures were maintained in an environmental chamber within the following conditions: a temperature of 24 ± 2 °C and continuous illumination of approximately 600 to 930 footcandles (6500 to 10 000 lux). Lighting was supplied by Duro-Test® Vita-Lite® fluorescent bulbs.
The fronds used to initiate the toxicity test with the test material were taken from a stock culture that had been transferred to fresh medium 2 days prior to testing.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

14 d

Test temperature:

23 - 26 °C

pH:

7.4 - 8.8

Nominal and measured concentrations:

6.3, 13, 25, 50 and 100 mg a.i./L (nominal)
5.2, 11, 21, 44 and 88 mg a.i./L (mean measured)

Details on test conditions:

TEST SYSTEM
- Environmental chamber used: yes. The test was conducted in an environmental chamber controlled to maintain a temperature of 24 ± 2 °C and continuous lighting with an intensity of 600 to 930 footcandles (6500 to 10 000 lux).
- Test vessel: Sterile 270-mL crystallising dishes were used as test vessels. One-hundred millilitres of the appropriate test solution was then placed in each replicate vessel. Each test vessel was covered with an inverted, sterile, glass Petri dish.
- Aeration: no
- Agitation: no
- No. of plants per vessel: five plants with three fronds each was aseptically introduced into each test vessel
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates):3
- No. of vessels per vehicle control (replicates): 3
Three sterile 270-mL crystallising dishes per treatment level and the controls were conditioned prior to use by rinsing with the appropriate test solution. Three control vessels, which contained 20X AAP medium and three solvent control vessels, which contained 0.10 mL/L of dimethylformamide and 20X AAP medium, were maintained under the same conditions as the treatment vessels but contained no test material.

GROWTH MEDIUM
- Standard medium used: yes. The culture medium used was 20X Algal Assay Procedure (AAP) medium. the composition of the 20X AAP medium was as follows: NaNO₃ (510 mg a.i./L); MgCl₂.6H₂O (243 mg a.i./L); CaCl₂.2H₂O (88.2 mg a.i./L); MgSO₄.7H₂O (294 mg a.i./L); K₂HPO₄.3H₂O (27.4 mg a.i./L); NaHCO₃ (300 mg a.i./L); H₃BO₃ (3.71 mg a.i./L); Na₂SeO₄ (0.0376 mg a.i./L); MnCl₂.4H2O (8.31 mg a.i./L); ZnCl₂ (0.0654 mg a.i./L); CoCl₂.6H2O (0.0286 mg a.i./L); CuCl₂.2H₂O (0.00024 mg a.i./L); Na₂MoO₄.2H₂O (0.145 mg a.i./L); FeCl₃.6H₂O 3.20 mg a.i./L); Na₂EDTA.2H₂O (6.00 mg a.i./L). If required, pH was adjusted to 7.5 ± 0.1 with 0.1 N NaOH or 0.1 N HCl

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: If required, pH was adjusted to 7.5 ± 0.1 with 0.1 N NaOH or 0.1 N HCl
- Photoperiod: continuous light
- Light intensity and quality: Light intensity of the test area ranged from 700 to 900 footcandles (7500 to 9700 lux). The photosynthetically active radiation (PAR) of the test area at test initiation ranged from 119 to 143 μE/m²/s.

EFFECT PARAMETERS MEASURED
On day 7 and at test termination (day 14), fronds were counted and observations were made. The test vessels were assigned new random positions within the environmental chamber after the day 7 observation interval.
At test termination (day 14), after frond density determinations were complete, the fronds were removed from each vessel, blotted dry and transferred to pre-weighed aluminium pans. Fronds were dried in an oven at 72 °C for three days prior to dry weight determination.

ENVIRONMENTAL PARAMETERS
Temperature was measured continuously with a Fisher Scientific minimum/maximum thermometer located in a flask of water adjacent to the test vessels within the environmental chamber. Temperature readings were recorded daily. Lighting was provided by Duro-Test® Vita-Lite® fluorescent bulbs. Light intensity was measured with a General Electric type 214 light meter at 0 hour and at each subsequent 24-hour interval during the exposure period.
The pH of the exposure solutions was measured at test initiation and test termination. The portion of the test solution remaining in the 500- or 2000-mL volumetric flasks after filling the test vessels was used for initial pH measurements. At test termination, after frond counts were completed, the three replicate vessels of the treatment levels and the control were respectively composited, and a portion of each composite solution was transferred to a 100-mL beaker for pH measurement. Test solution pH was measured with a Jenco 60 pH meter.

PRELIMINARY TESTING
A preliminary range-finding exposure was conducted at nominal test material concentrations of 0.0010, 0.010, 0.10, 1.0 and 10 mg a.i./L, a control and a solvent control. Two exposure vessels were established for each concentration and the controls. Following 14 days of exposure, the control and the solvent control both contained an average of 143 fronds/replicate (pooled control = 143 fronds/replicate). Frond densities in the 0.0010, 0.010, 0.10, 1.0 and 10 mg a.i./L treatment levels averaged 126, 822, 764, 856 and 786 fronds/replicate, respectively.
Fronds exposed to the 0.010, 0.10, 1.0 and 10 mg a.i./L treatment levels were observed to be smaller than the controls. Fronds in the remaining concentration (0.0010 mg a.i./L) and the controls were normal. Green algae, Pseudokirchneriella subcapitata, was observed in the control, solvent control and 0.0010 mg a.i./L solutions and was most likely responsible for the reduced frond numbers observed in these vessels. Based on these data, nominal concentrations of 6.3, 13, 25, 50 and 100 mg a.i./L were selected for the definitive exposure.

Reference substance (positive control):

no

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 88 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

88 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

> 88 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

88 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

44 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

frond number

Key result

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

> 88 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

frond number

Details on results:

FROND DENSITY
On day 7, fronds exposed to the treatment levels, the control and the solvent control were observed to be normal. The 7-day frond density in the control and solvent control solutions averaged 446 and 361 fronds per replicate, respectively (pooled control = 404). Frond production in the 5.2, 11, 21, 44 and 88 mg a.i./L treatment levels averaged 360, 348, 367, 414 and 372 fronds per replicate, respectively. Based on the results of Shapiro-Wilks' and Bartlett's Tests, this data set passed the requirements for normality and homogeneity of variance, therefore, Williams' Test was used to determine treatment-related effects. No significant reduction in frond density in any of the treatment levels as compared to the pooled control was detected.
On day 14, fronds exposed to the control, the solvent control, 5.2 and 11 mg a.i./L treatment levels were observed to be normal. Fronds exposed the 21 mg a.i./L treatment level were observed to have less root formation than the controls. Fronds exposed the 44 and 88 mg a.i./L treatment levels were observed to be slightly chlorotic and have less root formation than the controls. The 14-day frond density in the control and solvent control solutions averaged 863 and 793 fronds per replicate, respectively. A significant difference was detected (t-Test) between the control and solvent control data, therefore the 14-day treatment frond density data was compared to the solvent control data. Frond production in the 5.2, 11, 21, 44 and 88 mg a.i./L treatment levels averaged 776, 769, 761, 782 and 688 fronds per replicate, respectively. Based on the results of Shapiro- Wilks' and Bartlett's Tests, this data set passed the requirements for normality and homogeneity of variance, therefore, Williams' Test was used to determine treatment-related effects. Based on Williams’ Test, a significant reduction was detected in the 88 mg a.i./L treatment level relative to the solvent control data.

GROWTH RATE
The 7-day growth rate for the control and solvent control averaged 0.49 and 0.46 days^-1, respectively (pooled control = 0.47 days^-1). Frond biomass in the 5.2, 11, 21, 44 and 88 mg a.i./L treatment levels averaged 0.46, 0.45, 0.46, 0.48 and 0.46 days^-1, respectively. Based on the results of Shapiro-Wilks' and Bartlett's Tests, this data set passed the requirements for normality and homogeneity of variance, therefore, Williams' Test was used to determine treatment-related effects. No significant reduction in frond growth rate was detected in any of the treatment levels as compared to the pooled control.

BIOMASS
The 14-day biomass for the control and solvent control averaged 0.1442 and 0.1322 g, respectively (pooled control = 0.1382 g). Frond biomass in the 5.2, 11, 21, 44 and 88 mg a.i./L treatment levels averaged 0.1379, 0.1266, 0.1157, 0.1358 and 0.1214 g, respectively. Based on the results of Shapiro-Wilks' and Bartlett's Tests, this data set passed the requirements for normality and homogeneity of variance, therefore, Williams' Test was used to determine treatment-related effects. No significant reduction in frond biomass was detected in any of the treatment levels as compared to the pooled control.

Additional testing to further define the EC50 values was not performed since the highest nominal concentration tested was equivalent to the maximum test concentration required by the study guidelines.

Reported statistics and error estimates:

A t-test was used to compare the frond densities, growth rate and biomass of the control to the solvent control. If no significant difference was determined, control and solvent control data were pooled for further statistical analysis to determine treatment level effects. If a significant difference was detected, the treatment data were compared to the solvent control data.
The EC25 and EC50 values (concentrations of test material which caused 25 and 50 % reduction, respectively, in the 7- and 14-day frond density, 7-day growth rate EC50 and 14-day biomass) and the 95 % confidence limits, were determined by linear regression of response (percent reduction of frond density, growth rate or biomass as compared with the appropriate control) versus mean measured test concentration. If no concentration resulted in >25 or >50 % inhibition, the EC values were empirically estimated to be greater than the highest concentration tested.
The No-Observed-Effect Concentration (NOEC), the highest test concentration that caused no significant adverse effect on the 7- and 14-day frond density, 7-day growth rate and 14-day biomass when compared to the appropriate control, and Low-Observed-Effect Concentration (LOEC), were determined using Williams' Test. The data were first checked for normality using Shapiro-Wilks' Test and for homogeneity of variance using Bartlett's Test. If the data sets passed the test for homogeneity and normality, the Williams' Test was used to determine the NOEC. If the data did not pass the tests for homogeneity and normality, then Kruskal-Wallis' Test was used to determine the NOEC. All statistical determinations were made at the 95 % level of certainty, except in the case of Bartlett's and Shapiro-Wilks' Tests, where the 99 % level of certainty was applied.

Analytical Chemistry

Test solution samples analysed on day 0 and test termination (day 14) approximated the desired nominal concentrations. Mean measured concentrations ranged from 83 to 88 % of nominal concentrations and defined the treatment levels as 5.2, 11, 21, 44 and 88 mg a.i./L. Analysis of the quality control samples resulted in recoveries which were consistent with the pre-determined recovery range and ranged from 98.4 to 108 % (N = 6) of the nominal fortified levels (6.00 to 100 mg a.i./L). Based on the results of these analyses, it was established that the appropriate precision and quality control was maintained during the analysis of the exposure solutions.

Water Quality Parameters

The pH ranged from 7.4 to 8.8 throughout the exposure period. Continuous temperature monitoring established that the temperature ranged from 23 to 26 °C during the study period. Light intensity of the test area ranged from 700 to 900 footcandles (7500 to 9700 lux). The photosynthetically active radiation (PAR) of the test area at test initiation ranged from 119 to 143 μE/m²/s.

Table 1: Mean Frond Production After 7 and 14 Days of Exposure to the Test Material

Mean measured conc. (mg a.i./L)	Fronds/replicate
	Day 7	Day 14	14-day inhibition* (%)
Control	446	863	NA
Solvent control	361	793	NA
5.2	360	776	2.1
11	348	769	3.0
21	367	761**	4.0
44	414	782**~	1.3
88	372	688**~#	13

*Percent inhibition relative to the pooled control (404 fronds/replicate on day 7)

**Less root formation than the control

~Fronds were slightly chlorotic

#Significantly reduced as compared to the pooled control, based on Williams' Test.

Table 2: Mean Calculated Growth Rates After 7 Days of Exposure to the Test Material

Mean measured conc. (mg a.i./L)	Growth rate (days^-1)
	Day 7	7-day inhibition* (%)
Control	0.49	NA
Solvent control	0.46	NA
5.2	0.46	2
11	0.45	4
21	0.46	2
44	0.48	-2
88	0.46	2

*Percent inhibition relative to the pooled control (0.47 days^-1 on day 7)

Table 3: Mean Frond Dry Weight After 14 Days of Exposure to the Test Material

Mean measured conc. (mg a.i./L)	14-day dry weight (g)	Percent inhibition*
Control	0.1442	NA
Solvent control	0.1322	NA
Pooled control	0.1382	NA
5.2	0.1379	0
11	0.1266	8
21	0.1157	16
44	0.1358	2
88	0.1214	12

*Relative to the pooled control

Validity criteria fulfilled:

yes

Conclusions:

On day 7, no significant reduction in frond density in any of the treatment levels as compared to the pooled control was detected. Therefore, the 7-day NOEC and LOEC for frond density was determined to be 88 mg a.i./L and >88 mg a.i./L, respectively. The 7-day EC25 and EC50 values were empirically estimated to be >88 mg a.i./L, the highest concentration tested.
On day 14, based on Williams’ Test, a significant reduction in frond density was detected in the 88 mg a.i./L treatment level relative to the solvent control data. The NOEC and LOEC values were determined to be 44 mg a.i./L and 88 mg a.i./L, respectively. The 14-day EC25 and EC50 values were determined to be >88 mg a.i./L, the highest concentration tested.
No significant reduction in frond growth rate (p < 0.05) was detected in any of the treatment levels as compared to the pooled control. Therefore, the 7-day NOEC and LOEC for frond growth rate was determined to be 88 mg a.i./L and >88 mg a.i./L, respectively. The 7-day EC50 for frond growth was determined to be >88 mg a.i./L.
No significant reduction in frond biomass (p < 0.05) was detected in any of the treatment levels as compared to the pooled control. Therefore, the 14-day NOEC and LOEC for frond biomass was determined to be 88 mg a.i./L and >88 mg a.i./L, respectively. The 14-day EC25 and EC50 values for frond biomass was determined to be >88 mg a.i./L.

Executive summary:

The toxicity of the test material to the duckweed, Lemna gibba, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 221 and EPA OPP 122-2 and 123-2.

Based on the results of a preliminary test, nominal concentrations of test material of 6.3, 13, 25, 50 and 100 mg a.i./L were selected for the definitive exposure. A solvent (DMF) control and untreated control (20X Algal Assay Procedure (AAP) medium) were included. Three replicates were prepared for each concentration of test material, solvent and untreated control.

Approximately 30 minutes after the test solutions were prepared and added to the test vessels, an inoculum of five plants with three fronds each was aseptically introduced into each test vessel. Test vessels were then randomly placed, based on computer-generated random numbers, on a shelf within an environmental chamber.

On day 7 and at test termination (day 14), fronds were counted and observations were made. The test vessels were assigned new random positions within the environmental chamber after the day 7 observation interval. At test termination (day 14), after frond density determinations were complete, the fronds were removed from each vessel, blotted dry and transferred to pre-weighed aluminium pans. Fronds were dried in an oven at 72 °C for three days prior to dry weight determination.

Temperature was measured continuously with a minimum/maximum thermometer located in a flask of water adjacent to the test vessels within the environmental chamber. The pH of the exposure solutions was measured at test initiation and test termination.

Under the conditions of the study, no significant reduction in frond density in any of the treatment levels as compared to the pooled control was detected on day 7. Therefore, the 7-day NOEC and LOEC for frond density was determined to be 88 mg a.i./L and >88 mg a.i./L, respectively. The 7-day EC25 and EC50 values were empirically estimated to be >88 mg a.i./L, the highest concentration tested. On day 14, based on Williams’ Test, a significant reduction in frond density was detected in the 88 mg a.i./L treatment level relative to the solvent control data. The NOEC and LOEC values were determined to be 44 mg a.i./L and 88 mg a.i./L, respectively. The 14-day EC25 and EC50 values were determined to be >88 mg a.i./L, the highest concentration tested. No significant reduction in frond growth rate was detected in any of the treatment levels as compared to the pooled control. Therefore, the 7-day NOEC and LOEC for frond growth rate was determined to be 88 mg a.i./L and >88 mg a.i./L, respectively. The 7-day EC50 for frond growth was determined to be >88 mg a.i./L. No significant reduction in frond biomass was detected in any of the treatment levels as compared to the pooled control. Therefore, the 14-day NOEC and LOEC for frond biomass was determined to be 88 mg a.i./L and >88 mg a.i./L, respectively. The 14-day EC25 and EC50 values for frond biomass was determined to be >88 mg a.i./L.

Reference 2

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August 2012 to 18 December 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

other: draft guidance document of SETAC AMRAP (Aquatic Macrophyte Risk Assessment for Pesticides) working group 2

Deviations:

no

Principles of method if other than guideline:

The test was conducted following the proposed test method for the rooted aquatic macrophyte, Myriophyllum sp. The method is based partly on existing OECD guidelines 201 and 219, but includes modifications that reflect recent research and consultation on a number of key issues. The AMRAP test protocol is included in the test guideline program of the OECD.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

yes

Details on sampling:

The concentration of test material in the water and the sediment was assessed by chemical analysis. Water samples were taken from the freshly prepared test solutions at test start, from a pooled sample of the highest test concentration replicates on days 3 and 7, and from pooled samples of the test media at test end.
Since measured concentrations of test material in the water samples decreased by more than 30 % during the study, the sediment samples from day 14 were also analysed. Sediment from all treatment and control replicates was pooled per treatment.
All samples were frozen immediately after collection and were stored at -20 °C until analysis.

Vehicle:

no

Details on test solutions:

- Test media preparation
A stock solution of test material (1200 μg a.s./L) was prepared by adding 12.7 mg of test material into 10 L of Smart & Barko medium and stirring for 1 hour. This solution formed the highest test concentration (test concentration 5). Aliquots of 4000, 1600, 640, and 256 mL of the stock solution were added to 10 L of Smart & Barko medium to prepare the respective lower test concentrations. The control medium consisted of Smart & Barko medium only.

- Test media application
Following macrophyte preparation and pre-incubation Smart & Barko test medium was carefully poured into the beakers via a funnel so as not to disturb the sediment. Each vessel contained 1.8 L of test media at a minimum depth of 12 cm. The water level was marked. The water level was adjusted with distilled water to the marked level when evaporation loss during the test was more than 10 %.

Test organisms (species):

Myriophyllum spicatum

Details on test organisms:

TEST ORGANISM
- Common name: rooted aquatic macrophyte
- Species: Myriophyllum spicatum, Haloragaceae, Dicotyledonous
- Source: Institut für Gewässerschutz, MESOCOSM GmbH, Neu-Ulrichstein 5, 35315 Homberg (Ohm), Germany.
- Stock cultures: The stock cultures were grown under sterile conditions with medium containing sucrose

ACCLIMATION
The plants were grown under non-sterile standard conditions for at least one week prior to the seven day pre-incubation period of the test, in growth medium and sediment of the same type as used in the test (medium without sucrose) to deplete the plants of the sucrose.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

14 d

Test temperature:

The temperature of the growth room was 19.0 - 20.5 °C. The temperature of the test media was between 18.0 - 20.0 °C (mean 19.5 ± 0.5 °C).

pH:

The pH of the pooled controls at test start was 7.92 and between 10.10 and 10.32 for the individual control vessels at the end of the exposure period.

Dissolved oxygen:

Oxygen saturation of pooled control test media was 99.5 % at test start and between 120.2 -151.1 % at the end of the exposure period in individual control vessels.

Nominal and measured concentrations:

control, 30.7, 76.8, 192, 480 and 1200 μg a.s./L (nominal)

Details on test conditions:

TEST SYSTEM
- Test container: The test was conducted using 2 L glass beakers (approx. 24 cm high and 11 cm diameter) as replicate housing. Small plastic plant pots (approx. 9 cm diameter and 8 cm high, 350 mL, commercially available) were used for potting the plants into the sediment. The exposed sediment surface represented about 70 % of the cross-sectional area of the glass test vessels in which the pots were individually placed.

SEDIMENT
The following formulated sediment, based on the artificial soil used in OECD Guideline 219 was used:
(1) Standard sediment without additional nutrients:
(a) 5.02 % sphagnum peat (dry weight, according to 2 ± 0.5 % organic carbon in the final sediment) as close to pH 5.5 to 6.0 as possible. Peat in powder form, finely ground and air dried, was used.
(b) 20.0 % (dry weight) kaolin clay (kaolinite content above 30 %).
(c) 75.1 % (dry weight) quartz sand (fine sand).
(d) 14.3 L deionised water is added to obtain moisture of the final mixture of about 30 %.
(e) 50 g CaCO₃ was added to adjust the pH of the final mixture of the sediment to 7.0 ± 0.5.
(2) Nutrient supplemented sediment:
(f) An aqueous nutrient solution together with deionised water was used for sediment preparation to obtain a final moisture content of 30 %. One litre of a nutrient medium containing 6.66 g NH₄Cl and 15.4 g Na₃PO₄ x 12 H₂O were added to 33.3 kg sediment-mixture. Then 13.3 L deionised water was added (concentration in the final sediment: 200 mg Na₃PO₄/kg sediment and 200 mg NH₄Cl/kg sediment).
The constituents of the sediment were mixed homogenously. Water/nutrient solution (approx. 30 % w/w) was mixed thoroughly into this sediment. The moist sediment was stored for two days at room temperature to allow proper soaking of the peat and then frozen for 7 weeks at -20 °C.
Planting pots were prepared with a filter paper in the bottom of the pot to stop soil loss. A 1 cm layer of the standard sediment was added followed by a 4 cm layer of nutrient supplemented sediment. A second 1 cm layer of standard sediment was added followed by a fine layer (approx. 2 mm) of coarse quartz sand in order to reduce suspension of sediment into the water.

GROWTH MEDIUM
Smart & Barko medium was used as liquid growth medium. At test initiation, the pH of the medium was adjusted to a level of 7.5 - 8.0 to allow optimum plant growth (using 0.1 N NaOH).
The composition of the Smart & Barko medium was as follows:
CaCl₂.2H₂O (91.7 mg/L), MgSO₄.7H₂O (69.0 mg/L), NaHCO₃ (58.4 mg/L) and KHCO₃ (15.4 mg/L).

EXPERIMENTAL DESIGN
Five replicate test vessels were used for each treatment group, 10 replicate test vessels for the control group, and 10 replicates for the solvent control group. Each test vessel contained one sediment pot with three plant shoots. Each test vessel was randomly assigned to a different treatment group. A randomised design for the location of the test vessels in the growth chamber was required to minimise the influence of spatial differences in light intensity or temperature. A repositioning of the vessels in an impartial way was carried out after observations were made.

MACROPHYTE PREPARATION
> Pre-incubation
Healthy shoot apices 6 cm (± 0.5 cm) long were abscised from the stock culture plants, patted dry, and individually weighed. Five plants with a suitable weight 30 % range (range of 100 – 130 % based on the lowest used weight) were selected and potted 3 cm deep into the sediment in each pot. Shoots were then maintained for 7 days, in aquaria with enough tap water to cover the plants, in order to induce root development.
> Test initiation
After pre-incubation, the pots were relocated from the pre-incubation aquaria to individual 2 L glass beakers. The shoot length above the sediment was measured (day 0) using a ruler inside the test vessels. Two of the five plants per pot were removed leaving three individuals, uniform in size and appearance. Smart & Barko test medium was carefully poured into the beakers via a funnel so as not to disturb the sediment. Each vessel contained 1.8 L of test media at a minimum depth of 12 cm. The water level was marked. The water level was adjusted with distilled water to the marked level when evaporation loss during the test was more than 10 %.
To obtain the respective mean biomass data for day 0 (test start), five homogeneous plants from each of five additional pots were harvested at test initiation and the fresh and dry weights determined.

OTHER TEST CONDITIONS
- Light quality: Cool white fluorescent lighting (fluorescence tubes Philips Master TL-D 36 Watt/840-2A and 58 Watt/840-2A) was used
- Photoperiod: 16 hours of light / 8 hours of darkness
- Light intensity: 6000 - 8000 lux (measured in a photosynthetically active radiation, 400-700 nm)

EFFECT PARAMETERS MEASURED
The exposure period was 14 days. During this time, visual inspections of plant growth, morphological changes/symptoms, or any other unusual observations were recorded on days 5, 10, and 14. Shoot length (cm) was determined on days 7 and 14, from the level of the sediment, using a ruler inserted into the test vessel and stood against the straightened plant. If side shoots were present, their number and length were also measured. At test end (day 14), the plants were washed free of sediment before a visual qualitative assessment of the roots was made. Each plant was individually identified so that measurements at each time point could be determined and directly compared. Total fresh weight and dry weight (mg) after oven drying at 60 °C for 48 h was then determined.

VEHICLE CONTROL PERFORMED: yes

Reference substance (positive control):

no

Key result

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

188 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Remarks on result:

other: Parameter: fresh weight. 95 % CL 158 to 222 µg/L

Key result

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

363 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: Parameter: fresh weight. 95 % CL 300 to 445 µg/L

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

63.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Growth rate and yield

Remarks on result:

other: Parameters: total shoot length, fresh and dry weight

Details on results:

- Effects on shoot length: Shoot length inhibition effects were concentration dependent. Yield was inhibited by 9.4 – 85.2 % and the growth rate by 1.5 – 66.8 % compared to the control. The lowest test concentration (25.9 μg/L) had a growth enhancement effect on yield.
The number of side shoots was reduced as the concentration increased. In the three highest concentrations visual deformities of the shoot growth were observed.
- Effects on fresh weight: Fresh weight effects were concentration dependent. Yield was inhibited by 2.5 – 85.4 % and the growth rate by 0.8 – 69.4 % compared to the control. The lowest test concentration (25.9 μg/L) had a growth enhancement effect on both yield and growth rate.
- Effects on dry weight: Dry weight effects were concentration dependent. Yield was inhibited by 18.3 – 67.7 % and the growth rate by 6.8 – 43.1 % compared to the control.
- Effects on roots: A visual qualitative assessment of the roots at test end was made for additional information. A concentration dependent effect on the development of the root system was observed, with fewer roots per plant as the concentration increased.

Reported statistics and error estimates:

Statistical calculations were made for the results obtained for individual vessels (replicates), not for individual plants. Mean values were calculated for shoot length, fresh and dry weights per replicate. In the case of fresh and dry weights, the biomass at the start of the experiment was derived from a sample of 25 representative plants. Length of main shoot and length of side shoots were summed to give the total plant length.
From the measured variables yields (i.e. gain of weight or length) and growth rates (weight or length gain per time unit, on logarithmic scale) were calculated.
Yield variables were calculated according to the following formula: Yield = (Nt - N0)
Growth rates were calculated per replicate according to the following formula: Growth rate = (ln(Nt) - ln(N0)) / t
Where
N0 is fresh/dry weight or shoot length measured at the start of the exposure period t
Nt is fresh/dry weight or shoot length measured at the end of the exposure period t
t is time in days

The results for the parameters (growth rate and yield) were subjected to ANOVA. When treatment effects were detected a Williams' test was performed to derive the No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC). The EC50 values were calculated by probit analysis modified for continuous data. All statistical analyses were conducted by the computer program ToxRat Professional v.2.10.

Environmental Parameters

Light intensity, temperature, and pH values of the controls were in accordance with the recommended values. During the entire test period the light intensity was in the range of 6805 - 7432 lux and the growth room temperature 19.0 – 20.5 °C. The temperature of the test media was between 18.0 - 20.0 °C (mean 19.5 ± 0.5 °C). The pH of the pooled controls at test start was 7.92 and between 10.10 and 10.32 for the individual control vessels at the end of the exposure period. Oxygen saturation of pooled control test media was 99.5 % at test start, and between 120.2 - 151.1 % at the end of the exposure period in individual control vessels.

 

Analytical Chemistry

Measured concentrations in the water at test start were 31.9, 79.3, 198, 465, and 1236 μg/L (i.e. 97 - 104 % of nominal). The highest test concentration was 99.7and 90.7 % on days 3 and 7, respectively. By test end (day 14), the test concentrations were 21.0, 51.4, 124, 294, and 788 μg/L (i.e. 61.2 – 68.5 % of the nominal concentrations). As the measured concentrations were outside of the ± 20 % range of nominal, the geometric mean of the measured concentrations for each test concentration was calculated and used for the evaluation of the growth inhibition test. The geometric mean measured concentrations were 25.9, 63.9, 157, 370, and 987 μg/L (i.e. 77.1 – 84.4 % of nominal).

Since measured concentrations in the water samples decreased by more than 30 % during the study, the sediment samples were also analysed. The concentrations in the sediment at test end were < LOQ in the control and in the two lowest treatment levels. In the three highest test concentrations the sediment concentrations were 18.2, 38.5, and 110 μg/kg sediment dry matter.

Validity criteria fulfilled:

not applicable

Remarks:

Specific criteria for macrophyte growth tests have not yet been set. However, the study fulfilled the proposed validity criteria.

Conclusions:

In terms of EC50 values, the overall yield of each parameter was more affected than the growth rate. Fresh weight was the most sensitive parameter with EC50 values for yield and growth rate of 188 and 363 μg/L, respectively. In terms of the NOEC, all parameters were equally sensitive with significant inhibition relative to the control at concentrations higher than 63.9 μg/L (NOEC).

Executive summary:

The toxicity of the test material to sediment dwelling plants was investigated in a study which was conducted under GLP conditions and following the draft guidance document of SETAC AMRAP (Aquatic Macrophyte Risk Assessment for Pesticides) working group 2. The test was conducted following the proposed test method for the rooted aquatic macrophyte, Myriophyllum sp. The method is based partly on existing OECD guidelines 201 and 219, but includes modifications that reflect recent research and consultation on a number of key issues.

During the study, macrophytes were exposed to the following nominal concentrations of test material: control, 30.7, 76.8, 192, 480 and 1200 μg a.s./L. Five replicates per test concentration and ten replicates for the control were used. A replicate consisted of a single pot containing three plants.

The test material was directly dissolved in the growth medium. The concentration of test material in the water phase was assessed by chemical analysis (LCMS/ MS) at the start and at the end of the test for all treatment levels. The concentration in the highest test treatment was also assessed on days 3 and 7. Samples of the freshly prepared test solutions were taken at test start and all other samples were collected from pooled replicates per treatment. The minimum level of quantification (LOQ) for test material in water samples was 5.0 μg/L and in sediment samples 10.0 μg/kg.

The following growth parameters were measured: total shoot length, fresh weight, and dry weight. Plant total shoot length was recorded at test start and after 7 and 14 days. At test start, the fresh weight of the test plants and the dry weight of 25 representative additional plants were determined. At the end of the test all plants were harvested and washed. The root development was assessed qualitatively, as recommended in the AMRAP test protocol. Thereafter, fresh and dry weights were recorded. Effective concentrations (EC50) were calculated for the yield and the growth rate of each of the measured parameters.

Shoot length inhibition effects were concentration dependent. Yield was inhibited by 9.4 – 85.2 % and the growth rate by 1.5 – 66.8 % compared to the control. The lowest test concentration (25.9 μg/L) had a growth enhancement effect on yield. The number of side shoots was reduced as the concentration increased. In the three highest concentrations visual deformities of the shoot growth were observed. Fresh weight effects were concentration dependent. Yield was inhibited by 2.5 – 85.4 % and the growth rate by 0.8 – 69.4 % compared to the control. The lowest test concentration (25.9 μg/L) had a growth enhancement effect on both yield and growth rate. Dry weight effects were concentration dependent. Yield was inhibited by 18.3 – 67.7 % and the growth rate by 6.8 – 43.1 % compared to the control.

In terms of EC50 values, the overall yield of each parameter was more affected than the growth rate. Fresh weight was the most sensitive parameter with EC50 values for yield and growth rate of 188 and 363 μg/L, respectively. In terms of the NOEC, all parameters were equally sensitive with significant inhibition relative to the control at concentrations higher than 63.9 μg/L (NOEC).

Description of key information

7 day ErC50 > 88 mg a.i./L; 7 day NOEC (growth rate) = 88 mg a.i./L; 14 day EbC50 > 88 mg a.i./L; 14 day NOEC (biomass) = 88 mg a.i./L; 14 day EC50 (frond number) > 88 mg a.i./L; 14 day NOEC (frond number) = 44 mg a.i./L, OECD 221 and EPA OPP 122-2 and 123-2, Hoberg (2003)
14 day EC50 (growth rate) = 363 µg/L, 14 day EC50 (yield) = 188 µg/L, 14 day NOEC = 63.9 µg/L, Myriophyllum spicatum, draft guidance document of SETAC AMRAP (Aquatic Macrophyte Risk Assessment for Pesticides) working group 2 (method based partly on existing OECD guidelines 201 and 219), Wenzel (2014)

Key value for chemical safety assessment

EC50 for freshwater plants:

363 µg/L

EC10 or NOEC for freshwater plants:

63.9 µg/L

Additional information

Two studies investigating the toxicity of the substance to aquatic plants are available. Both studies were conducted under GLP conditions and in accordance with standardised guidelines. Both studies were assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997).

The toxicity of the test material to the duckweed, Lemna gibba, was investigated in a study conducted in accordance with the standardised guidelines OECD 221 and EPA OPP 122-2 and 123-2.

Based on the results of a preliminary test, nominal concentrations of test material of 6.3, 13, 25, 50 and 100 mg a.i./L were selected for the definitive exposure. A solvent (DMF) control and untreated control (20X Algal Assay Procedure (AAP) medium) were included. Three replicates were prepared for each concentration of test material, solvent and untreated control.

Approximately 30 minutes after the test solutions were prepared and added to the test vessels, an inoculum of five plants with three fronds each was aseptically introduced into each test vessel. Test vessels were then randomly placed, based on computer-generated random numbers, on a shelf within an environmental chamber.

On day 7 and at test termination (day 14), fronds were counted and observations were made. The test vessels were assigned new random positions within the environmental chamber after the day 7 observation interval. At test termination (day 14), after frond density determinations were complete, the fronds were removed from each vessel, blotted dry and transferred to pre-weighed aluminium pans. Fronds were dried in an oven at 72 °C for three days prior to dry weight determination.

Temperature was measured continuously with a minimum/maximum thermometer located in a flask of water adjacent to the test vessels within the environmental chamber. The pH of the exposure solutions was measured at test initiation and test termination.

Under the conditions of the study, no significant reduction in frond density in any of the treatment levels as compared to the pooled control was detected on day 7. Therefore, the 7-day NOEC and LOEC for frond density was determined to be 88 mg a.i./L and >88 mg a.i./L, respectively. The 7-day EC25 and EC50 values were empirically estimated to be >88 mg a.i./L, the highest concentration tested. On day 14, based on Williams’ Test, a significant reduction in frond density was detected in the 88 mg a.i./L treatment level relative to the solvent control data. The NOEC and LOEC values were determined to be 44 mg a.i./L and 88 mg a.i./L, respectively. The 14-day EC25 and EC50 values were determined to be >88 mg a.i./L, the highest concentration tested. No significant reduction in frond growth rate was detected in any of the treatment levels as compared to the pooled control. Therefore, the 7-day NOEC and LOEC for frond growth rate was determined to be 88 mg a.i./L and >88 mg a.i./L, respectively. The 7-day EC50 for frond growth was determined to be >88 mg a.i./L. No significant reduction in frond biomass was detected in any of the treatment levels as compared to the pooled control. Therefore, the 14-day NOEC and LOEC for frond biomass was determined to be 88 mg a.i./L and >88 mg a.i./L, respectively. The 14-day EC25 and EC50 values for frond biomass was determined to be >88 mg a.i./L.

In the study reported by Wenzel (2014) the toxicity of the test material to sediment dwelling plants was investigated in a study which was conducted in line with the draft guidance document of SETAC AMRAP (Aquatic Macrophyte Risk Assessment for Pesticides) working group 2. The test was conducted following the proposed test method for the rooted aquatic macrophyte, Myriophyllum sp. The method is based partly on existing OECD guidelines 201 and 219, but includes modifications that reflect recent research and consultation on a number of key issues.

During the study, macrophytes were exposed to the following nominal concentrations of test material: control, 30.7, 76.8, 192, 480 and 1200 μg a.s./L. Five replicates per test concentration and ten replicates for the control were used. A replicate consisted of a single pot containing three plants.

The test material was directly dissolved in the growth medium. The concentration of test material in the water phase was assessed by chemical analysis (LCMS/ MS) at the start and at the end of the test for all treatment levels. The concentration in the highest test treatment was also assessed on days 3 and 7. Samples of the freshly prepared test solutions were taken at test start and all other samples were collected from pooled replicates per treatment. The minimum level of quantification (LOQ) for test material in water samples was 5.0 μg/L and in sediment samples 10.0 μg/kg.

The following growth parameters were measured: total shoot length, fresh weight, and dry weight. Plant total shoot length was recorded at test start and after 7 and 14 days. At test start, the fresh weight of the test plants and the dry weight of 25 representative additional plants were determined. At the end of the test all plants were harvested and washed. The root development was assessed qualitatively, as recommended in the AMRAP test protocol. Thereafter, fresh and dry weights were recorded. Effective concentrations (EC50) were calculated for the yield and the growth rate of each of the measured parameters.

Shoot length inhibition effects were concentration dependent. Yield was inhibited by 9.4 – 85.2 % and the growth rate by 1.5 – 66.8 % compared to the control. The lowest test concentration (25.9 μg/L) had a growth enhancement effect on yield. The number of side shoots was reduced as the concentration increased. In the three highest concentrations visual deformities of the shoot growth were observed. Fresh weight effects were concentration dependent. Yield was inhibited by 2.5 – 85.4 % and the growth rate by 0.8 – 69.4 % compared to the control. The lowest test concentration (25.9 μg/L) had a growth enhancement effect on both yield and growth rate. Dry weight effects were concentration dependent. Yield was inhibited by 18.3 – 67.7 % and the growth rate by 6.8 – 43.1 % compared to the control.

In terms of EC50 values, the overall yield of each parameter was more affected than the growth rate. Fresh weight was the most sensitive parameter with EC50 values for yield and growth rate of 188 and 363 μg/L, respectively. In terms of the NOEC, all parameters were equally sensitive with significant inhibition relative to the control at concentrations higher than 63.9 μg/L (NOEC).