mecoprop-P (ISO); (R)-2-(4-chloro-2-methylphenoxy)propionic acid

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:

supporting study

Study period:

10 July 2014 to 08 August 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 239 (Water-Sediment Myriophyllum spicatum Toxicity Test)

Version / remarks:

2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD 219 - Sediment-Water Chironomid toxicity Using Spiked Water

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

One sample from the freshly prepared stock solution and duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test.
For the determination of the stability of the test material under the test conditions and of the maintenance of the test material concentrations during the test period, duplicate samples were taken at the end of the test from all test media and the control of the actual test by pouring together the contents of each treatment.
- Storage of Samples: All samples were separated into overlying water, pore water and sediment directly after sampling and stored in a freezer (≤ - 10 °C), protected from light until analysis was performed.
- Fortified Samples: Approximately 50 mg test material were diluted with 50 mL acetonitrile to obtain a stock solution of 1 g test material/L which was stirred for 30 minutes. Appropriate amounts of the stock solution were diluted with test water to obtain fortified samples at concentrations of 5, 50 and 1 250 μg test material/L.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Before the exposure period a concentrated stock solution of 20 mg/L was prepared by dispersing 27.4 mg test material in 1 370 mL test water by intense stirring for 20 minutes. Each treatment group was spiked with a defined volume of the stock solution in order to obtain the desired test concentrations.
The test media was prepared just before introduction of the aquatic plants (= start of the test).
- Evidence of undissolved material: The appearance of the test material in the test media was visually determined on each observation day in all test concentrations.

Test organisms (species):

Myriophyllum spicatum

Details on test organisms:

TEST ORGANISM
Holding conditions of sterile stock culture: The plants of the stock culture (shoot segments with two whorls without a shoot tip) were maintained in modified Andrews’ medium containing 3 % sucrose under sterile conditions for 36 days to initiate the development of side shoots. They are cultured at 3 470 lux and a temperature range of 20 °C.
Preparation of test organisms: New initiated side shoots were washed for 30 minutes in deionised water to remove the whole culture medium. For approximately 6 days the plants are maintained in Smart and Barko medium until the test start at a light intensity of 8 380 lux (16 hours photoperiod daily) and temperature conditions of 23°C to remove all plant stored sucrose.
The side shoots, which were used in the static growth inhibition test, had a length of 6 ± 1 cm before introducing them into the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

14 d

Remarks on exposure duration:

Duration of prior rooting phase: 7 days

Test temperature:

19 - 21 °C

pH:

7.9 at pre-rooting phase
8.0 – 8.1 at test start
7.9 – 8.8 on day 8
7.8 – 9.8 at test end

Dissolved oxygen:

7.6 – 8.6 mg/L at test start
7.5 – 9.6 mg/L on day 8
8.1 – 16.4 mg/L at test end

Nominal and measured concentrations:

1 000, 316, 100, 31.7 and 10 μg test material/L and a control, corresponding to 474, 150, 47.4, 15.0, 4.74 μg a.i./L.

Details on test conditions:

- Introduction of Sediment: The sediment was filled into standard planting pots of a diameter which just fit into the glass vessels (the sediment covered a minimum of 70 % of the vessel bottom surface). First, a filter paper was put on the bottom of the planting pot. Afterwards the pots was filled with the sediment.
- Introduction of Plants: Healthy shoot apices from healthy culture plants (without any flowers) were clipped off at a length of 6 cm (± 1 cm). Five shoot tips were planted into each pot containing the sediment with two nodes covered into the sediment. In order to induce root development they were kept prior to the test start for 7 days in test water.
- Introduction of Test Water: After potting the plants into the sediment the pots were set into the test beakers and the test water without any test material concentration was added very carefully in order to avoid any disturbance of the sediment. The level of test water was marked at the outside of the test beaker. If necessary, the water levels will be filled up with deionised water during the study to the original starting volume to prevent concentration of the test material. Water levels should not change by more than 10 %.
- Exposure: After the pre-culture period on Day 0, two of the five plants in each test beaker were removed to leave with three largely homogeneously (size and appearance) performing individuals.
The aged test water will be removed completely and replaced by the test media of the desired test concentrations or the control, respectively.
The day of application of the test material was designated as Day 0 (= start of the test).

TEST SYSTEM
- Incubation chamber used: Controlled environment room.
- Test vessel: Small plant pots (approx. 8.5 cm diameter, 7 cm high and with a volume of approx. 400 mL) were used as containers for potting the plants into the sediment.
Test beakers of 2 000 mL volume (approx. 11.5 cm diameter, 24 cm high) with approximately 1 800 mL test medium were used to provide an overlaying water depth of minimum 12 cm.
The sediment surface coverage was > 70 % of the test beakers surface.
- Material, size, headspace, fill volume: Glass beaker
- No. of vessels per concentration: Five replicates per test concentration, each with one plant.
- No. of vessels per control: Ten replicates, each with one plant.

SEDIMENT USED
- Origin: Sediment was prepared according to OECD test guideline 219, based on dry weight:
5 % sphagnum moss peat: As close to pH 5.0 - 6.0 as possible, no visible plant remains, finely ground and air dried;
75 % quartz sand (grain size: > 50 % of the particles should be in the range of 50 - 200 μm);
20 % kaolin clay (kaolinite content ≥ 30 %);
CaCO3, of chemically pure quality was added to adjust the pH of the final mixture of the sediment to 6.9.
Aqueous nutrient medium containing Na3PO4 and NH4Cl was added to obtain a moisture content of the final mixture in a range of 30 - 50 %.
The organic carbon content of the final mixture was 2.185 %.
The peat was air dried and ground to a fine powder. Then the necessary amount of peat powder was mixed with the other constituents (sand and kaolinite clay) and an aqueous nutrient medium, containing Na3PO4 and NH4Cl, to obtain homogeneous sediment with moisture content of 36.65 % of the dry weight of the sediment. The concentration of both nutrients was 200 mg/kg dry sediment.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Analytical grade salts were added at the following nominal concentrations in deionised water (conductivity <5 μScm^-1): 58.4 mg/L NaHCO3, 69.0 mg/L MgSO4 × 7 H2O, 15.4 mg/L KHCO3 and 91.7 mg/L CaCl2 × 2 H2O.
- Intervals of water quality measurement: The temperature was measured daily in a test vessel filled with water and incubated under the same conditions as the test vessels.
The pH-values were measured in the test media of all test concentrations and the control at test start, in the middle of the test and at test end.
The oxygen-values were measured in the test media of all test concentrations and the control at test start, in the middle of the test and at test end.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No, the pH was 7.9.The test water was prepared 2 and 6 days before introducing of the plants to allow pH to stabilise.
- Photoperiod: 16 h light: 8 h dark
- Light intensity and quality: 8 380 lux (mean value) with a range of during pre-rooting phase; 8 818 lux (mean value) with a range of 8 250 - 9 890 lux during exposure. The illumination was achieved by fluorescent tubes, installed above the test vessels. Measurements were performed at test start at 6 places distributed over the experimental area at the surface of the test media. Differences in light intensity over the test area did not exceed ± 15 %.

EFFECT PARAMETERS MEASURED:
- Shoot length: At test start (Day 0) the shoot length above the sediment and the length of any side shoot was measured from all plants which were used in the test. At test end the shoot length and the length of any side shoot were measured.
- Shoot weight (fresh / dry): For the determination of the wet and dry weight five additional pots each with two plants were prepared and kept simultaneously with the test beakers in the pre-culture period. After the pre-culture period these five pots were harvested and the plant biomass was determined from every pot to obtain the wet weight of the test start. For the weighing the plants were carefully blotted to remove the remaining test medium. Subsequently the dry weight of these plants was determined by drying them at 60 °C for at least two hours.
At test end the fresh and dry weight of every test plant was measured in the same manner as at the test start.
- Other: Any sublethal symptoms e.g. chlorosis or necrosis were recorded once during the test (Day 8) and at the end of the test. At test end the existence of roots and their appearance were also recorded.
- Determination of Growth Inhibition: The inhibition of growth was determined from shoot length, wet weight and dry weight. For these parameters, growth rate and yield were calculated.
Determination of Yield (y) Reduction: The reduction of yield was determined from:

%Iy = ((bC - bT) / bC) x 100

Where:
%Iy = Percent reduction in yield
bC = Final shoot length / biomass minus starting shoot length / biomass for the control group
bT = Final shoot length / biomass minus starting shoot length / biomass in the treatment group

Determination of Average Specific Growth Rate:

μi-j = (1n(Nj) – 1n(Ni)) / t

Where:
μi-j = Average specific growth rate from time i to j
Ni = The shoot length / the biomass at time i
Nj = The shoot length / the biomass at time j
t = Time period from i to j

Percentage inhibition of growth rate r (Ir):

%Ir = ((μC – μT) / μC) x 100

Where:
%Ir = Percentage inhibition in average specific growth rate
μC = Mean growth rate of the control
μT = Mean growth rate of test concentration i

RANGE-FINDING STUDY
Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions. The pre-experiments were not performed in compliance with the GLP-Regulations

Reference substance (positive control):

no

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

280 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

shoot length

Remarks on result:

other: 95 % confidence limits 199 - 418 µg/L

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

82.1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence limits 29.1 - 208 µg/L

Key result

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

69.4 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence limits 23.1 - 180 µg/L

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

10 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

shoot length

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

10 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

31.7 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

31.7 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

shoot length

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

31.7 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

14 d

Dose descriptor:

EC50

Remarks:

Yield

Effect conc.:

58.4 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

shoot length

Remarks on result:

other: 95 % confidence limits 44.3 - 76.2 µg/L

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

47 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95 % confidence limits 15.9 - 107 µg/L

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

22.4 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95 % confidence limits 11.0 - 36.6 µg/L

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

10 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

shoot length

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

< 10 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

< 10 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

31.7 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

shoot length

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

10 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

10 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

- Any visual signs of phytotoxicity: Sublethal effects were recorded for all test concentration groups except of the lowest. Beginning with 31.7 mg test material/L all plants had shorter and distorted leaves in comparison to the control after 8 and 14 days of exposure. At 100 - 1 000 μg test material/L the leaves were laid to the stem and they were shorter as well after eight and 14 days of exposure.
All plants developed roots. Beginning with 31.7 μg test material/L only a few roots were developed and they were shorter.
Side shoots frequently occurred in the control and the lower concentration ranges but their numbers decreased with increasing test material concentrations.
- Any observations that might cause a difference between measured and nominal values: There were no remarkable observations on the appearance of the test media.

- Growth inhibition: After 14 days the mean control shoot length was 73.8 cm compared to 64.2, 53.8, 23.9, 21.3 and 18.3 cm in the test material concentrations of 10, 31.7, 100, 316 and 1 000 μg test material/L.
There were significant reductions in yield in all test concentrations after 14 days of exposure. Therefore the NOEyC value after 14 days of exposure was determined to be 10 μg test material/L with a corresponding LOEyC value of 31.7 μg test material/L. The 14-day EyC50 was determined to be 58.4 μg test material/L. The EyC10 for shoot length after 14 days was calculated to be smaller than the lowest test concentration of 10 μg test material/L.

Statistical significant inhibitions for growth rate were determined for all treatment groups except of the lowest (10 μg/L) after 14 days. Therefore the NOErC value after 14 days of exposure was determined to be 10 μg test material/L with a corresponding LOErC value of 31.7 μg test material/L. The 14-day ErC50 was determined to be 280 μg test material/L. The ErC10 for shoot length after 14 days was calculated to be smaller than the lowest test concentration of 10 μg test material/L.

According to the statistical evaluation for yield wet weight a significant reduction occurred at all test material concentrations which resulted in a 14-day NOEC of < 10 μg test material/L with a corresponding 14-day LOEC of 10.0 μg test material/L. For the growth rate the NOEC is determined to be 10 μg test material/L with a corresponding LOEC of 31.7 μg test material/L directly from the raw data.
The EyC50 for wet weight was calculated to be 22.4 μg test material/L. The corresponding EyC20 and EyC10 were both smaller than the lowest test concentration of 10 μg test material/L. The ErC50 for wet weight was calculated to be 82.1 μg test material/L. The corresponding ErC20 and ErC10 were both smaller than the lowest test concentration of 10 μg test material/L.
According to the statistical evaluation for yield dry weight a significant reduction occurred at all test material concentrations which resulted in a 14-day NOEC of < 10 μg test material/L with a corresponding 14-day LOEC of 10 μg test material/L. The statistical evaluation for growth rate based on dry weight showed a significant reduction at the three highest test material concentrations which resulted in a 14-day NOEC of 31.7 μg test material/L with a corresponding 14-day LOEC of 100 μg test material/L.
The EyC50 for dry weight was calculated to be 47.0 μg test material/L. The corresponding EyC20 and EyC10 were both smaller than the lowest test concentration of 10 μg test material/L. The ErC50 for wet weight was calculated to be 69.4 μg test material/L. The corresponding ErC20 and ErC10 were both smaller than the lowest test concentration of 10 μg test material/L.

Reported statistics and error estimates:

The EC10/20/50 (the concentrations of the test item corresponding to 50, 20 and 10 % inhibition of growth rate (shoot length, wet weight and dry weight) and yield (shoot length, wet weight and dry weight) compared to the control), and their 95 % confidence limits were calculated by Probit analysis.
For the determination of the LOEyC and NOEyC values significant differences at the test concentrations compared to the control values were tested by the Bonferroni-Welch t-test (shoot length) and the Williams t-test (wet weight and dry weight). Based on the data set some inhibition values are regarded as not significant, although the statistical evaluation considers them as significant. Inhibition rates below 25 % and the absence of sublethal effects leads to this decision.
For the determination of the LOErC and NOErC values significant differences at the test concentrations compared to the control values were tested by the Williams t-test (shoot length), Bonferroni-Welch t-test (wet weight) and the Williams t-test (dry weight). The statistical evaluation based on the mean values per replicate.
The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat® Solutions GmbH.

Summary of Biological Results

Parameter		Yield (Total Shoot Length) [μg/L]	Growth Rate (Total Shoot Length) [μg/L]	Yield (Wet Weight) [μg/L]	Growth Rate (Wet Weight) [μg/L]	Yield (Dry Weight) [μg/L]	Growth Rate (Dry Weight) [μg/L]
EC50	Test material	58.4 (44 – 76.2)	280 (199 – 418)	22.4 (11.0 – 36.6)	82.1 (29.1 – 208)	47.0 ( 15.9 – 107)	69.4 (23.1 – 180)
	Active ingredient	27.7 (21.0 – 36.1)	133 (94.8 – 198)	10.6 (5.22 – 17.3)	38.9 (13.8 – 98.6)	22.3 (7.52 – 50.8)	32.9 (10.9 – 85.3)
EC20	Test material	12.9 (7.45 – 18.9)	26.4 (13.0 – 42.4)	< 10	< 10	< 10	< 10
	Active ingredient	6.13 (3.53 – 6.18)	12.5 (6.18 – 20.1)	< 4.74 (n.d.)	< 4.74 (n.d.)	< 4.74 (n.d.)	< 4.74 (n.d.)
EC10	Test material	< 10 (n.d.)	< 10 (<10 – 15.1)	< 10 (n.d.)	< 10 (n.d.)	< 10 (n.d.)	< 10 (< 10 – 12.6)
	Active ingredient	< 4.74 (n.d.)	< 4.74 (n.d.)	< 4.74 (n.d.)	< 4.74 (n.d.)	< 4.74 (n.d.)	< 4.74 (n.d.)
14-Day NOEC	Test material	10.0	10.0	< 10	10.0	< 10	31.7
	Active ingredient	4.74	4.74	< 4.74	4.74	< 4.74	15.0
14-Day LOEC	Test material	31.7	31.7	10.0	31.7	10.0	10.0
	Active ingredient	15.0	15.0	4.74	15.0	4.74	47.4

Values refer to nominal test concentrations.

Values in parentheses are 95 % confidence limits.

n.d. Could not be determined.

Yields y and Growth Rates μ (Based on Shoot Length) and Percentage Inhibition of y and μ (Based on Shoot Length) During the Test Period of 14 days

Nominal Concentration [μg Test Material/L]	Yields y (total shoot length) [cm] and % Inhibition of y 0 – 14 Days		Growth Rate μ (Total Shoot Length) [1/Day] and % Inhibition of μ 0 – 14 Days
	y	%	μ	%
Control	66.8	-	0.166	-
10	57.6	13.8(*)	0.162	2.1
31.7	45.7	31.6*	0.136	17.9*
100	16.7	74.9*	0.085	48.6*
316	13.9	79.2*	0.076	54.1*
1 000	11.1	83.4*	0.066	59.9*

m: Mean value.

s: Standard deviation.

- % inhibition: Increase in growth relative to that of control.

* Mean value significantly different from the control (tested with Welch t-test (Yield) and Williams t-test (Growth rate), α = 0.05, one-sided).

(*) Values are indicated as significant by the statistical evaluation. Based on the biological data the effect is regarded as not significant.

 

Percentage Inhibition of y and μ (All Based on Wet weights) After 14 days of Exposure

Nominal Concentration [μg Test Material/L]	Yields y (Wet Weight) and % Inhibition of y (After 14 Days)		Growth Rate μ (Wet Weight) and % Inhibition of μ (After 14 Days)
	y (mg)	%	μ [1/Day]	%
Control	1195.9	-	0.122	-
10	921.6	31.3*	0.102	16.8(*)
31.7	827.0	55.9*	0.079	35.3*
100	158.6	86.7*	0.029	76.7*
316	193.8	83.8*	0.038	68.6*
1 000	275.4	77.0*	0.050	58.8*

- % inhibition: Increase in growth relative to that of control.

* Mean value significantly different from the control (tested with William t-test, α = 0.05, one-sided (yield) and Bonferroni-Welch test, α = 0.05, one-sided (growth rate)).

(*) Values are indicated as significant by the statistical evaluation. Based on the biological data the effect is regarded as not significant.

 

Percentage Inhibition of y and μ (All Based Dry weights) After 14 days of Exposure

Nominal Concentration [μg Test Material/L]	Yields y (Dry Weight) and % Inhibition of y (After 14 Days)		Growth Rate μ (Dry Weight) and % Inhibition of μ (After 14 Days)
	y (mg)	%	μ [1/Day]	%
Control	71.2	-	0.049	-
10	50.6	28.9*	0.038	22.0
31.7	49.4	30.6*	0.036	26.8*
100	10.4	85.4*	0.007	86.3*
316	15.4	78.4*	0.013	73.5*
1 000	10.6	85.1*	0.009	81.5*

- % inhibition: Increase in growth relative to that of control.

* Mean value significantly different from the control (tested with Williams t-test, α = 0.05, one-sided).

Validity Criteria of the Study

- Increase in shoot length of control plants: Compared to initial values (100 %) control values after 14 days were 1010 %.

- Increase in fresh weight length of control plants: Compared to initial values (100 %) control values after 14 days were 564 %

- Coefficient of variation for yield (based on wet weight of control plants): The coefficient of variation of yield wet weight was 23.1 %

- Sublethal effects in control plants: Control plants did not show any sign of sublethal effects and were visibly free from contamination by other organisms such as algae and/or bacterial film.

Summary of Analytical Results

Sample [μg Test Material/L)	% of Nominal*	RSD [%]	n
Control	n.a	n.a	4
10	101	12	4
31.7	105	9	4
100	105	7	4
316	98	8	4
1 000	100	8	4

* Mean value of all measured samples per treatment group.

RSD: Relative standard deviation per treatment group.

n: Number of analysed samples.

n.a.: Not applicable.

Validity Criteria of the Analytical Part

Specificity:  No interference of total peak area for the target analyte was found.

Linearity: Calibration Range: 0.5 to 75 μg test material/L.

Linearity of Response: Correlation of peak area of different standard solutions with their corresponding concentrations, using a linear regression.

Regression Coefficient: r = 0.9999

Calibration Curve: y = 22621 * x - 552

Repeatability of Injection:  The relative standard deviation of repeated injection of one standard solution is 1 %.

Accuracy and Precision:  Mean Recovery Rates in the Fortified Samples: 101 % (n = 15, RSD 3%).

The values found for the precision (RSD) and for the accuracy (mean recovery rate) are acceptable.

Conclusion:  The validity criteria for the analytical method have been met.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 14-day EyC50 was calculated to be 58.4, 22.4 and 47.0 μg test material/L for total shoot length, wet weight and dry weight, respectively.
The 14-day ErC50 was calculated to be 280, 82.1 and 69.4 μg test material/L for shoot length, wet weight and dry weight, respectively.
The 14-day NOEyC and the LOEyC were determined to be 10 and 31.7 μg test material/L for shoot length, < 10 and 10 μg test material/L for wet weight and dry weight, respectively.
The 14-day NOErC and the LOErC were determined to be 10 and 31.7 μg test material/L for shoot length and wet weight and 31.7 and 100 μg test material/L for dry weight, respectively.

Executive summary:

The toxicity of the test material to the aquatic plant Myriophyllum spicatum was assessed according to OECD Test Guideline 239 and in compliance with GLP.

Plants of were exposed in a static test to various concentrations of the test material under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 14 days.

This study encompassed 6 treatment groups (5 dose rates of the test material and a control) with five replicates per test concentration and ten replicates for the control.

After a pre-rooting phase of 7 days, one plant per replicate was incubated for 14 days under static conditions. The shoot length was determined at test start and day 14. Sub-lethal parameters were assessed at test start, once during the test (day 8) and at test end. At test end fresh and dry weight of each replicate was determined.

The samples collected at start and after 14 days were analysed via HPLC-MS/MS method.

Yield and growth rate based on main shoot length, wet and dry weight

At the start of the test 109 % of the nominal test concentration was found (average of all test concentrations). After 14 days test duration, 94 % of the nominal value was determined (average of all test concentrations). During the test the plants were exposed to a mean of 102 % of nominal. Therefore, all reported results refer to nominal concentrations.

Under the conditions of the study the 14-day EyC50 was calculated to be 58.4, 22.4 and 47.0 μg test material/L for total shoot length, wet weight and dry weight, respectively.

The 14-day ErC50 was calculated to be 280, 82.1 and 69.4 μg test material/L for shoot length, wet weight and dry weight, respectively.

The 14-day NOEyC and the LOEyC were determined to be 10 and 31.7 μg test material/L for shoot length, < 10 and 10 μg test material/L for wet weight and dry weight, respectively.

The 14-day NOErC and the LOErC were determined to be 10 and 31.7 μg test material/L for shoot length and wet weight and 31.7 and 100 μg test material/L for dry weight, respectively.

Reference 2

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 November 2013 to 19 December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 239 (Water-Sediment Myriophyllum spicatum Toxicity Test)

Version / remarks:

OECD Draft Guideline: Water-Sediment Myriophyllum sp Toxicity Test based on Draft AMRAP Method: Growth Inhibition Test for the Rooted Aquatic Macrophyte, Myriophyllum sp. Submitted to OECD for Evaluation, 22 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analytical data are required by the OECD guidelines to demonstrate the correct concentration of the test material (representative samples).
Duplicate analytical samples were taken. One of each sample was analysed and the other stored as a backup.
Samples of the overlying water were taken from the test vessels for all concentration levels and control and analysed for the active ingredient at test start and end.
At test termination on day 14 wet sediment was sampled from the test vessels for all concentration levels and control and analysis of the active ingredient were performed on the sediment.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The necessary amount of the test material for preparing the stock solution was weighed on a weighing scoop and transferred to a volumetric flask. Test medium was added up to the bench mark and the solution was homogenised by shaking. The lower test concentrations were made by dilution of the stock solution with test medium. Same amounts of the respective stock solution and dilutions were applied to the respective test vessels containing test medium.

Test organisms (species):

Myriophyllum spicatum

Details on test organisms:

TEST ORGANISM
- Source: Myriophyllum spicatum plants have been maintained under laboratory conditions at Eurofins Agroscience Services EcoChem GmbH since November 2010. The cultures obtained from Umweltbundesamt Berlin, Germany were based on a culture of the Landesanstalt für Gewässerkunde Koblenz, Germany.
- Method of cultivation: M. spicatum is cultivated under sterile conditions submersed in a modified, aqueous Andrews medium containing sucrose. This laboratory stock culture is used to provide uniform plants throughout the year, eliminating seasonal variation in plant quality and contamination by other species (e.g. algae). The stock culture plants were held under the same environmental conditions as used in the test.

ACCLIMATION
- Culturing media and conditions: 15 days prior to test initiation plant were cultivated under the test condition in Smart and Barko medium. Eight days prior to test initiation, submerged apical shoots of the same size (5 cm in length and without side shoots) were planted in an aquarium (60 cm length x 30 cm width x 30 cm height) in an artificial sterilised sediment overlaid with Smart and Barko medium under the same temperature, light, and water quality conditions as used during the exposure of the plants in the test. Shoots were anchored in an upright position using metal rings and were maintained under controlled environment conditions. Approx. 120 shoots were cultivated in this manner in order to generate sufficient uniform material for use in the test. Only plants of the same size (e.g. ± 10 - 20 % of mean shoot length) were used for the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

14 d

Test temperature:

19.2 ± 0.6 °C

pH:

8.06 ± 0.65

Dissolved oxygen:

115 ± 17 %

Nominal and measured concentrations:

1.91, 6.10, 19.5, 62.5 and 200 μg/L test material and control. This is equivalent to 0.917, 2.93, 9.37, 30.0 and 96.1 μg/L active ingredient.

Details on test conditions:

- Test System: Plants were grown in a static water-sediment system using artificial sterilised sediment overlaid with Smart and Barko medium under the same conditions as used in the pre-culture. The study was conducted in 2 L glass-beakers measuring approx. 12 cm in diameter and 24 cm height. Only one shoot per test vessel was planted. The volume of added water was recorded, and the level marked on the outside of the test vessels.
- Preparation of the Test Vessels: 350 g of moist sediment was transferred to the test vessels. The surface was overlaid with a thin layer of washed quartz sand to minimise displacement of the sediment when the medium was added. Afterwards the test vessels were filled carefully with growth medium (1.5 L) to a depth of 14 cm. Two days after preparation of the test vessels and before application one rooted apical shoot per vessel was planted carefully, ensuring the plant was rooted into the sediment. Shortly afterwards, application of the test item was performed and mixed in with gentle stirring.

TEST SYSTEM
- Incubation chamber used: Test vessels were maintained in a controlled environment. Test vessels were arranged in a completely randomised design and were rerandomised several times during the study.
- No. of vessels per concentration: Five for each test material group.
- No. of vessels per control: Ten replicates were used for the control.

GROWTH MEDIUM
- Standard medium used: Liquid growth medium for aseptic Myriophyllum spicatum stock culture. Modified Andrews solution containing sucrose for culturing of Myriophyllum spicatum apical shoots. The sediment was overlaid with Smart and Barko growth medium.
- Detailed composition if non-standard medium was used:
Growth media: 3.73 mg/L KCl, 80.8 mg/L KNO3, 188.8 mg/L Ca(NO3)2 x 4 H2O, 98.6 mg/L MgSO4 x 7 H2O, 0.169 mg/L MnSO4 x H2O, 0.15 mg/L ZnSO4 x 7 H2O, 0.155 mg/L H3BO3, 0.012 mg/L CuSO4 x 5 H2O, 0.0037 mg/L (NH4)6Mo7O24 x 4 H2O, 27.2 mg/L KH2PO4, 2.78 mg/L FeSO4 x 7 H2O, 3.72 mg/L Na2EDTA, 30 g/L Sucrose (Added to make a modified ANDREWS solution). This medium was autoclaved before use.
Smart and Barko medium for the pre-culture and testing of rooted Myriophyllum spicatum shoots: 91.7 mg/L CaCl2 x 2 H2O, 69.0 mg/L MgSO4 x 7 H2O, 58.4 mg/L NaHCO3, 15.4 mg/L KHCO3. pH (air equilibrium) approximately 7.9.

SEDIMENT USED
- Origin: The following artificial sediment, based on the artificial soil used in OECD Guideline 219 and recommended in the AMRAP protocol, was used (percentages based on dry weight). The artificial sediment was prepared using the following materials:
4 % sphagnum peat (approximately pH 5.5 - 6.0; no visible plant remains, finely ground, air dried); commercial product 'Floratorf' obtained from Floragard Vertriebs GmbH für Gartenbau, 26138 Oldenburg, Germany;
20 % kaolin clay (kaolinite content above 30 %); obtained from Erbslöh GmbH & Co.KG, 65359 Geisenheim, Germany;
75 - 76 % quartz sand (fine sand with more than 50 % of the particles between 50 and 200 microns); obtained from Goldmann Baustoffe GmbH & Co. KG Güterbahnhof 10, 75177 Pforzheim, Germany;
Approximately 0.2 % calcium carbonate, precipitated extra pure, to adjust the sediment pH to 7.0 ± 0.5 at the start of the test before adding the test material; precipitated extra pure (Merck, Darmstadt, Germany);
Organic carbon content of the final mixture should be 2 % (± 0.5 %) and was adjusted by the use of appropriate amounts of peat and sand;
200 mg of ammonium chloride and sodium phosphate per L wet sediment.
The dry constituents were blended in the correct proportions and mixed thoroughly in an electric mixer. The dry sediment was sterilised in a heating chamber at 110 °C for at least 2 hours prior to use to minimise algal contamination of the test systems.


TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: Water temperature, pH and dissolved oxygen content were recorded on days 0, 7 and 14. Light intensity on the water surface was measured once during the test. The depth of water in each test vessel was measured at the intervals specified above and topped up with deionised water as necessary, if evaporation had occurred.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: The test material had no influence on the pH-value of the test solutions.
- Photoperiod: 16-h day-length.
- Light intensity and quality: Warm and/or cool white fluorescent lighting (approx. 130 – 160 μE*m-2*s-1 at the water surface).

EFFECT PARAMETERS MEASURED: Assessments of plant growth were made on days 0, 7 and 14 during the test.
On day 0 fifteen additional plants, representative of those used in the test, were selected from the available plant material. The plants were blotted dry prior to assessment of plant fresh weight and shoot length. The plants were placed separately in labelled glass beakers and dried at 60 °C for > 48 hours. The weight of the dry plant samples was recorded.
On day 14 plants were harvested from each treatment group for assessment of biomass (plant fresh weight and plant dry weight), shoot length and number and length of side shoots. In addition observations on shoot and root development (e.g. necrosis, deformation) were documented.
Specific growth rate was calculated as:

μi-j = (1n(Nj) – 1n(Ni)) / t

Where:
μi-j = Average specific growth rate from time i to j
Ni = Total shoot length or the biomass at time i
Nj = Total shoot length or the biomass at time j
t = Time period from i to j

and % inhibition for specific growth rate was calculated as:

%Ir = ((μC – μT) / μC) x 100

Where:
%Ir = Percentage inhibition in average specific growth rate
μC = Mean growth rate of the control
μT = Mean growth rate of the respective test item concentration

For the test material concentrations and control, mean values for yield were calculated. The mean percent inhibition in yield was calculated for each treatment group as follows:

%Iy = ((bC – bT) / bC) x 100

Where:
%Iy = Percent reduction in yield
bC = Final total shoot length minus starting total shoot length for the control group, or final biomass minus starting biomass for the control group
bT = Final total shoot length minus starting total shoot length in the test item treatment group, or final biomass minus biomass in the test material treatment group

The mean doubling time (Td) of the culture for the control and treatment were calculated using the following formula:

Td = ln2/μ

Where μ is the mean of the growth rates.

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: Yes, based on the results of a non-GLP range-finding test.

Reference substance (positive control):

no

Key result

Duration:

14 d

Dose descriptor:

EC50

Remarks:

Growth

Effect conc.:

26.9 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

shoot length

Duration:

14 d

Dose descriptor:

EC50

Remarks:

Growth

Effect conc.:

53.3 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

14 d

Dose descriptor:

EC50

Remarks:

Growth

Effect conc.:

> 96.1 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: No effect > 50 % could be observed, therefore the EC50 was estimated to be estimated to be based on active ingredient content.

Duration:

14 d

Dose descriptor:

EC50

Remarks:

Yield

Effect conc.:

9.41 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

shoot length

Duration:

14 d

Dose descriptor:

EC50

Remarks:

Yield

Effect conc.:

12 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

14 d

Dose descriptor:

EC50

Remarks:

Yield

Effect conc.:

> 96.1 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: No effect > 50 % could be observed, therefore the EC50 was estimated to be estimated to be based on active ingredient content.

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

0.971 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

shoot length

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

0.971 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

96.1 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

The control plants showed uniform growth over the test period of 14 days, with strongly growing side shoots. Over 14 days, the mean total shoot length increased more than 5.8-fold, fresh weight biomass increased more than 5.4-fold, and mean dry weight biomass increased more than 2.3-fold.
The mean control growth rate based on shoot length, fresh weight and dry weight was 0.1250, 0.1199 and 0.0601/day respectively, which is equivalent to a mean doubling time of 5.5, 5.8 and 11.5 days respectively. The coefficient of variation (C.V.) for control growth based on shoot length, fresh weight and dry weight was 9, 14 and 28 % respectively. The mean control yield (and C.V.) based on shoot length was 32.3 cm (C.V. = 19 %), for fresh weight yield was 1.0375 g (C.V. = 28 %), and for dry weight yield was 0.0528 g (C.V. = 40 %). Therefore, mean control growth rates and variability were considered acceptable.
Development of the main shoot length was determined after 7 and 14 days. The plants in the control and the test material concentration up to 19.5 μg/L showed a similar uniform growth over the test period of 14 days. At 62.5 and 200 μg/L plants showed a reduced growth in main shoot length during the test period of 14 day compared to the control.
Furthermore, all effects on plant growth (e.g. deformation, chlorosis, necrosis) during the test were documented; visible effects of the test material on shoot development were apparent after 7 and 14 days.
After 14 days the mean control total shoot length was 39.0 cm. This compared to 34.6, 32.7, 31.7, 15.8 and 11.1 cm in the test material concentrations of 1.91, 6.10, 19.5, 62.5 and 200 μg test material/L, respectively. Upon closer inspection it was apparent that the reduction in total shoot length was due a reduction of main shoot length as well a reduction in the number and length of side shoots formed during the exposure period.
The percentage inhibition of growth rate in comparison to the control after 14 days are also given. No statistically significant inhibition was determined up to and including 19.5 μg/L after 14 days of exposure. Therefore the NOEC value after 14 days of exposure was determined to be 19.5 μg test material/L with a corresponding LOEC value of 62.5 μg test material/L. The 14-day ErC50 was determined to be 56.1 μg test material/L.
The percentage inhibitions of the yields in comparison to the control after 14 days are also given. No statistically significant inhibition was determined up to and including 1.91 μg/L after 14 days of exposure. Therefore the NOEC value after 14 days of exposure was determined to 1.91 μg test material/L with a corresponding LOEC value of 6.10 μg test material/L. The 14-day EyC50 was determined to be 19.6 μg test material/L.
After 14 days the mean control fresh weight was 1.2690 g. This compared to 1.1173, 0.9988, 0.9833, 0.6151 and 0.4700 g in the test material concentrations of 1.91, 6.10, 19.5, 62.5 and 200 μg test material/L, respectively.
Statistical comparison to the control identified a significant reduction in 14-day fresh weight (growth rate) for concentrations ≥ 62.5 μg test material/L which resulted in a 14-day NOEC of 19.5 μg test material /L for growth rate with a corresponding 14-day LOEC of 62.5 μg test material /L. The 14-day ErC50 for the parameter fresh weight was determined to be 111 μg test material/L.
Statistical comparison to the control identified a significant reduction in 14-day fresh weight (yield) for concentrations ≥ 6.10 μg test material/L which resulted in a 14-day NOEC of 1.91 μg test material/L with a corresponding 14-day LOEC of 6.10 μg test material/L was observed. The 14-day EyC50 for the parameter fresh weight was determined to be 24.9 μg test material/L.
After 14 days the mean control dry weight was 0.0912 g. This compared to 0.0857, 0.0775, 0.0821, 0.0862 and 0.0737 g in the test material concentrations of 1.91, 6.10, 19.5, 62.5 and 200 μg test material/L, respectively.
Statistical comparison to the control identified no significant reduction in 14-day dry weight (growth rate and yield) at any concentration level. Since no significant effects on dry weight could be observed, the NOEC for growth rate and yield based on biomass (dry weight) was estimated to be 200 μg/L. A 14-day LOEC could not be determined. Since there was less than 50 % inhibition of growth at any test concentration, the 14-day ErC50 and EyC50 for the parameter dry weight were estimated to be > 200 μg test material/L.
- Any observations that might cause a difference between measured and nominal values: All environmental parameters measured were within the range recommended in the AMRAP Draft Method.

Reported statistics and error estimates:

Data were used to calculate the following parameters for each plant on day 14: Growth rate for total shoot length, yield for total shoot length, growth rate for plant fresh weight, yield for plant fresh weight, growth rate for plant dry weight, yield for plant dry weight. For each of these parameters EC50 values were calculated and in addition the NOEC and LOEC were determined where possible.
LOEC/NOEC Determination: All data were subjected to ANOVA. A test for normality of the data was carried out by calculating the Shapiro-Wilk’s statistic. For homogeneity of variances across treatment groups a Bartlett’s or Levene’s test was performed. Since data were normally distributed and variance was homogeneous a Dunnett’s test was performed. (SAS® Proprietary Software 9.3).
ECx Estimation: The ECx (yield and growth rate) was calculated where possible using Probit analysis. Only concentrations within a clear dose response were used for calculations.

Summary of Biological Results Based on Nominal Concentrations of the Test Material and Total Shoot Length

Parameter	Growth Rate (Total Shoot Length in cm) [μg/L] (μg/L Active Ingredient Equivalent*)	Yield (Total Shoot Length in cm) [μg/L] (μg/L Active Ingredient Equivalent*)
14-Day EC50	56.1 (26.9)	19.6 (9.41)
95 % Conf. Limits	40.9 – 80.8	14.8 – 26.5
14-Day EC20	8.41 (4.04)	3.04 (1.46)
95 % Conf. Limits	5.82 – 11.5	2.05 – 4.21
14-Day EC10	3.12 (1.50)	1.15 (0.552)
95 % Conf. Limits	1.90 – 4.61	0.677 – 1.74
14-Day NOEC	19.5 (9.37)	1.91 (0.917)
14-Day LOEC	62.5 (30.0)	6.10 (2.93)

*Based on the active ingredient content of the test material and density (1.252) as given in the Certificate of Analysis.

 

Summary of Biological Results based on Nominal Concentrations of the Test Material and Fresh Weight

Parameter	Growth Rate (Fresh Weight in g) [μg/L] (μg/L Active Ingredient Equivalent*)	Yield (Fresh Weight in g) [μg/L] (μg/L Active Ingredient Equivalent*)
14-Day EC50	111 (53.3)	24.9 (12.0)
95 % Conf. Limits	69.7 – 205	17.5 - 37.0
14-Day EC20	8.46 (4.06)	2.11 (1.01)
95 % Conf. Limits	5.23 – 12.7	1.19 – 3.28
14-Day EC10	2.20 (1. 60)	0.582 (0.265)
95 % Conf. Limits	1.07 – 3.73	0.255 – 1.05
14-Day NOEC	19.5 (9.37)	1.91 (0.917)
14-Day LOEC	62.5 (30.0)	6.10 (2.93)

*Based on the active ingredient content of the test material and density (1.252) as given in the Certificate of Analysis.

 

Summary of Biological Results based on Nominal Concentrations of the Test Material and Dry Weight

Parameter	Growth Rate (Dry Weight in g) [μg/L] (μg/L Active Ingredient Equivalent*)	Yield (Dry Weight in g) [μg/L] (μg/L Active Ingredient Equivalent*)
14-Day EC50	>200† (>96.1†)	>200† (>96.1†)
95 % Conf. Limits	-	-
14-Day EC20	-	-
95 % Conf. Limits	-	-
14-Day EC10	-	-
95 % Conf. Limits	-	-
14-Day NOEC	200 (96.1)	200 (96.1)
14-Day LOEC	n.d.	n.d.

* Based on active substance content of 601.4 g/L and density (1.252) as given in the Certificate of Analysis.

† No effect > 50 % could be observed, therefore the EC50 was estimated to be >200 μg/L test material or >96.1 μg/L acid equivalent.

- Not calculable.

n.d. Not determined.

Mean Total Shoot Length Including Side Shoots (cm)

Nominal Concentration Test Material [μg/L]	0† Days After Application	14 Days After Application	Yield [cm]	Reduction in Yield [%]	Growth Rate [1/Day]	Reduction in Growth Rate [%]
Control	6.7	39.0	32.3	-	0.1250	-
1.91	6.7	34.6	27.9	13.6	0.1171	6.3
6.10	6.7	32.7	26.0*	19.5*	0.1129	9.7
19.5	6.7	31.7	25.0*	22.6*	0.1107	11.4
62.5	6.7	15.8	9.1*	71.8*	0.0594*	52.5*
200	6.7	11.1	4.4*	86.4*	0.0341*	72.7*

† Based on 15 additional plants, representative of those used in the test.

* Significantly different reduction compared to the control.

 

Mean Total Plant Fresh Weight (g)

Nominal Concentration Test Material [μg/L]	0† Days After Application	14 Days After Application	Yield [g]	Reduction in Yield [%]	Growth Rate [1/Day]	Reduction in Growth Rate [%]
Control	0.2315	1.269	1.0375	-	0.1129	-
1.91	0.2315	1.1173	0.8858	14.6	0.1120	6.6
6.10	0.2315	0.9988	0.7673*	26.0*	0.1043	13.0
19.5	0.2315	0.9833	0.7518*	27.5*	0.1021	14.8
62.5	0.2315	0.6151	0.3536*	63.0*	0.0691*	42.4*
200	0.2315	0.4700	0.2385*	77.0*	0.0493*	58.9*

† Based on 15 additional plants, representative of those used in the test.

* Significantly different reduction compared to the control.

 

Observations on Shoot and Root Development Over the Test Period

Nominal Concentration Test Material [μg/L]	Shoot Development†	Root Development‡
Control	No effects	No effects
1.91	No effects	No effects
6.10	No effects	No effects
19.5	No effects	No effects
62.5	Hanging leaves, deformation of shoot	Reduced number
200	Hanging leaves, deformation of shoot	Reduced number and length of roots

†Observations on day 7 and 14.

‡Observations on day 14.

Validity Criteria

This study can be regarded as valid, since the OECD test guideline was adopted 26 September 2014 and results are in the acceptable range mentioned in the recent guideline.

- The total shoot length and plant fresh weight in control plants double during the exposure phase of the test and control plants showed no visual symptoms of chlorosis.

- The mean coefficient of variation for growth rate and yield based on measurements of total plant fresh weight (i.e. from test initiation to test termination) in the control cultures did not exceed 35 %.

Analytical Results

Recovery of the Active Ingredient From the Test Material Spiked into Test Medium

Test Material Fortification Level (μg/L)	Nominal (μg/L)	Found (μg/L)	Recovery (%)	Mean Recovery ± RSD (%)
0.2	0.0961	0.0846	88	91 ± 2
		00888	92
		0.0868	90
		0.0894	93
		0.0866	90
250	120	117	98	96 ± 2
		113	94
		117	98
		114	95
		114	95

RSD: Relative standard deviation.

 

Recovery of the Active Ingredient From the Analytical Reference Material Spiked into Sediment

Fortification Level* (mg/kg)	Found (mg/kg)	Recovery (%)	Mean Recovery ± RSD (%)
0.005	0.00469	94	93 ± 5
	0.00480	96
	0.00482	96
	0.00457	91
	0.00430	86
0.1	0.0768	79	93 ± 9
	0.0972	97
	0.0948	95
	0.101	101
	0.0908	91

RSD: Relative standard deviation.

* Purity considered.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study there was no effect > 50 % observed for the ErC50 and EyC50 for biomass dry weight, therefore the EC50 was estimated to be > 96.1 μg a.i./L.
The 14 day ErC50 was 26.9 μg/L, 53.5 μg/L and > 0.96.1 μg/L based on shoot length, biomass (fresh weight) and biomass (dry weight) respectively.
The 14 day EyC50 was 9.41 μg/L, 12.0 μg/L and > 96.1 μg/L, based on shoot length, biomass (fresh weight) and biomass (dry weight) respectively.
The 14 day NOEC was 0.971 μg/L for shoot length and biomass (fresh weight) and 96.1 μg/L based on biomass (dry weight).

Executive summary:

The toxicity of the test material to aquatic plants was assessed according to OECD Draft Guideline: Water-Sediment Myriophyllum sp Toxicity Test based on Draft AMRAP Method: Growth Inhibition Test for the Rooted Aquatic Macrophyte, Myriophyllum sp. Submitted to OECD for Evaluation, 22 July 2013 and in compliance with GLP.

Five replicates per test material concentration and ten replicates for the control were used. The duration of the test was 14 days. The test was performed under static test conditions. The nominal concentrations of the test material during the test were 1.91, 6.10, 19.5, 62.5 and 200 μg/L and control. This is equivalent to 0.917, 2.93, 9.37, 30.0 and 96.1 μg/L active ingredient. The test material was spiked to the water.

Test material concentrations in the definitive test were verified by analysis of the active ingredient at all concentration levels by analysing the overlying water at test start and test end and wet sediment at test termination on day 14.

On day 14 plants were harvested from each treatment group for assessment of shoot length, plant fresh weight, plant dry weight and number and length of side shoots. Additionally, the main shoot length was measured by use of a ruler on days 0, 7 and 14 during the test.

Endpoints reported are the EC50 for yield (EyC50) and growth rate (ErC50) based on the increase in total shoot length and biomass respectively after 14 days of exposure. The NOEC and LOEC for yield and growth rate were also determined. Temperature, pH and oxygen saturation [%] of the test solutions, measured after 0, 7 and 14 days, are reported.

The measured concentration of the test material based on the active ingredient content in the test vessels at test start ranged between 102 and 111 % of nominal in the overlaying water. The mean measured content for all concentrations at test start was 107 % of nominal for the active ingredient. After 14 days mean active ingredient concentrations in the overlaying water were below 95 % of nominal. As the mean contents of active ingredient were between 80 and 120 % of nominal at test start all toxicological endpoints were evaluated using nominal concentrations of the test material.

In the sediment amounts above the LOQ were only detected at the two highest concentration levels of 62.5 and 200 μg/L test material. The measured content in the sediment at day 14 was 7 % of nominal.

Since the guideline allows a mean coefficient of variation in the control cultures of 35 % EC20 and EC10 values should be treated with caution. It is likely that an increase or decrease of 10 - 20 % is disproportionately affected by the control variability. Consequently, statistical extrapolation of values which are below the lowest test concentration level or estimated from a flat dose response are likely to be unrealistic due to the natural variation between single plants. Total shoot length was found to be more sensitive than biomass (fresh and dry weight) for the EC50.

Under the conditions of the study there was no effect > 50 % observed for the ErC50 and EyC50 for biomass dry weight, therefore the EC50 was estimated to be > 96.1 μg a.i./L.

The 14-day ErC50 was 26.9 μg/L, 53.5 μg/L and > 0.96.1 μg/L based on shoot length, biomass (fresh weight) and biomass (dry weight) respectively.

The 14-day EyC50 was 9.41 μg/L, 12.0 μg/L and > 96.1 μg/L, based on shoot length, biomass (fresh weight) and biomass (dry weight) respectively.

The 14-day NOEC was 0.971 μg/L for shoot length and biomass (fresh weight) and 96.1 μg/L based on biomass (dry weight).

Description of key information

Gonsior (2015)

Under the conditions of the study there was no effect > 50 % observed for the ErC50 and  EyC50 for biomass dry weight, therefore the EC50 was estimated to be > 96.1 μg a.i./L.

The 14 day ErC50 was 26.9 μg/L, 53.5 μg/L and > 0.96.1 μg/L based on shoot length, biomass (fresh weight) and biomass (dry weight) respectively.

The 14 day EyC50 was 9.41 μg/L, 12.0 μg/L and > 96.1 μg/L, based on shoot length, biomass (fresh weight) and biomass (dry weight) respectively.

The 14 day NOEC was 0.971 μg/L for shoot length and biomass (fresh weight) and 96.1 μg/L based on biomass (dry weight).

 

Seeland-Fremer & Mosch (2015)

Under the conditions of the study the 14-day EyC50 was calculated to be 58.4, 22.4 and 47.0 μg test material/L for total shoot length, wet weight and dry weight, respectively.

The 14-day ErC50 was calculated to be 280, 82.1 and 69.4 μg test material/L for shoot length, wet weight and dry weight, respectively.

The 14-day NOEyC and the LOEyC were determined to be 10 and 31.7 μg test material/L for shoot length, < 10 and 10 μg test material/L for wet weight and dry weight, respectively.

The 14-day NOErC and the LOErC were determined to be 10 and 31.7 μg test material/L for shoot length and wet weight and 31.7 and 100 μg test material/L for dry weight, respectively.

Additional information

Gonsior (2015)

The toxicity of the test material to aquatic plants was assessed according to OECD Draft Guideline: Water-Sediment Myriophyllum sp Toxicity Test based on Draft AMRAP Method: Growth Inhibition Test for the Rooted Aquatic Macrophyte, Myriophyllum sp. Submitted to OECD for Evaluation, 22 July 2013 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Five replicates per test material concentration and ten replicates for the control were used. The duration of the test was 14 days. The test was performed under static test conditions. The nominal concentrations of the test material during the test were 1.91, 6.10, 19.5, 62.5 and 200 μg/L and control. This is equivalent to 0.917, 2.93, 9.37, 30.0 and 96.1 μg/L active ingredient. The test material was spiked to the water.

Test material concentrations in the definitive test were verified by analysis of the active ingredient at all concentration levels by analysing the overlying water at test start and test end and wet sediment at test termination on day 14.

On day 14 plants were harvested from each treatment group for assessment of shoot length, plant fresh weight, plant dry weight and number and length of side shoots. Additionally, the main shoot length was measured by use of a ruler on days 0, 7 and 14 during the test.

Endpoints reported are the EC50 for yield (EyC50) and growth rate (ErC50) based on the increase in total shoot length and biomass respectively after 14 days of exposure. The NOEC and LOEC for yield and growth rate were also determined. Temperature, pH and oxygen saturation [%] of the test solutions, measured after 0, 7 and 14 days, are reported.

The measured concentration of the test material based on the active ingredient content in the test vessels at test start ranged between 102 and 111 % of nominal in the overlaying water. The mean measured content for all concentrations at test start was 107 % of nominal for the active ingredient. After 14 days mean active ingredient concentrations in the overlaying water were below 95 % of nominal. As the mean contents of active ingredient were between 80 and 120 % of nominal at test start all toxicological endpoints were evaluated using nominal concentrations of the test material.

In the sediment amounts above the LOQ were only detected at the two highest concentration levels of 62.5 and 200 μg/L test material. The measured content in the sediment at day 14 was 7 % of nominal.

Since the guideline allows a mean coefficient of variation in the control cultures of 35 % EC20 and EC10 values should be treated with caution. It is likely that an increase or decrease of 10 - 20 % is disproportionately affected by the control variability. Consequently, statistical extrapolation of values which are below the lowest test concentration level or estimated from a flat dose response are likely to be unrealistic due to the natural variation between single plants. Total shoot length was found to be more sensitive than biomass (fresh and dry weight) for the EC50.

Under the conditions of the study there was no effect > 50 % observed for the ErC50 and EyC50 for biomass dry weight, therefore the EC50 was estimated to be > 96.1 μg a.i./L.

The 14-day ErC50 was 26.9 μg/L, 53.5 μg/L and > 0.96.1 μg/L based on shoot length, biomass (fresh weight) and biomass (dry weight) respectively.

The 14-day EyC50 was 9.41 μg/L, 12.0 μg/L and > 96.1 μg/L, based on shoot length, biomass (fresh weight) and biomass (dry weight) respectively.

The 14-day NOEC was 0.971 μg/L for shoot length and biomass (fresh weight) and 96.1 μg/L based on biomass (dry weight).

Seeland-Fremer & Mosch (2015)

The toxicity of the test material to the aquatic plant Myriophyllum spicatum was assessed according to OECD Test Guideline 239 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Plants of were exposed in a static test to various concentrations of the test material under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 14 days.

This study encompassed 6 treatment groups (5 dose rates of the test material and a control) with five replicates per test concentration and ten replicates for the control.

After a pre-rooting phase of 7 days, one plant per replicate was incubated for 14 days under static conditions. The shoot length was determined at test start and day 14. Sublethal parameters were assessed at test start, once during the test (day 8) and at test end. At test end fresh and dry weight of each replicate was determined.

The samples collected at start and after 14 days were analysed via HPLC-MS/MS method.

Yield and growth rate based on main shoot length, wet and dry weight

At the start of the test 109% of the nominal test concentration was found (average of all test concentrations). After 14 days test duration, 94 % of the nominal value was determined (average of all test concentrations). During the test the plants were exposed to a mean of 102 % of nominal. Therefore, all reported results refer to nominal concentrations.

Under the conditions of the study the 14-day EyC50 was calculated to be 58.4, 22.4 and 47.0 μg test material/L for total shoot length, wet weight and dry weight, respectively.

The 14-day ErC50 was calculated to be 280, 82.1 and 69.4 μg test material/L for shoot length, wet weight and dry weight, respectively.

The 14-day NOEyC and the LOEyC were determined to be 10 and 31.7 μg test material/L for shoot length, < 10 and 10 μg test material/L for wet weight and dry weight, respectively.

The 14-day NOErC and the LOErC were determined to be 10 and 31.7 μg test material/L for shoot length and wet weight and 31.7 and 100 μg test material/L for dry weight, respectively.