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Ecotoxicological information

Toxicity to aquatic plants other than algae

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Reference
Endpoint:
toxicity to aquatic plants other than algae
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 2016-11-15 to 2016-11-25, with the definitive exposure phase from 2016-11-16 to 2016-11-23.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
All test item concentrations and the control were analytically verified via HPLC-DAD at the start (0 day, fresh medium) and at the end of the exposure (7 days, old medium).
Details on test solutions:
Stock solution
10.0 mg test item/L were prepared with dilution water.

Dispersion treatment
The stock solution was stirred for 23 hours (1100 rpm, room temperature).

Test concentrations
Based on the results of a preliminary range finding test 5 concentrations were tested with a dilution factor of 2: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L.

Control
Six replicates (without test item) were tested under the same test conditions as the test vessels.
Test organisms (species):
Lemna gibba
Details on test organisms:
Test organism
Duckweed, Lemna gibba, Lemnaceae, Arales, Arecidae, Monocotyledonae
Young, rapidly growing plants without visible lesions or discolouration (chlorosis) were used for the test.

Reason for the selection of the test organism
According to the guideline, Lemna gibba is a suitable species because it is a representative of temperate areas commonly used for toxicity tests.

Origin
EUROFINS-GAB GMBH, Eutinger Str. 24, 75223 Niefern-Öschelbronn, Germany

Date of receipt
2008-02-26

Cultivation at test facility
The species is cultured in the test facility. Density is kept low to prevent conglomerates of plants on the surface. At least once per week, plants are transferred to freshly prepared growth medium. Growth media and culturing vessels are autoclaved before use to enable the breeding of axenic cultures.

Breeding vessels
Crystallisation dishes of glass, vol. 900 mL, filled with ca. 500 mL growth medium, covered with glass tops

Medium
20X-AAP-medium (Algal Assay Procedure medium),
pH-value 7.5 ± 0.1, see dilution water

Temperature 24 ± 2 °C

Light regime
Continuous fluorescent light, 1100 – 4440 lux

Acclimatization of the test system
The test system (the test organism) was held for 7 days under test conditions to acclimatize. These acclimatized plants were used in the test.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Hardness:
not measured
Dissolved oxygen:
not measured
Salinity:
not measured
Conductivity:
not measured
Nominal and measured concentrations:
Nominal: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L
Geometric mean measured test item concentrations: 0.440 – 1.09 – 2.39 – 5.05 – 10.8 mg/L
Details on test conditions:
Test method
Static procedure

Test duration
7 days

Replicates
3 replicates per concentration level, 6 for the control.

Test vessels/test volumes
Crystallisation dishes with a volume of 500 mL, covered with glass tops and filled with 200 mL test solution were used in the test. The test vessels were placed on a black non-reflective surface to avoid stray light.


Dilution water
20X-AAP-medium according to the guideline.

Composition of Dilution water
Component Concentration in stock solution [g/L] Concentration in prepared medium [mg/L]
NaNO3 26 510
MgCl2  6 H2O 12 240
CaCl2  2 H2O 4.4 90
MgSO4  7 H2O 15 290
K2HPO4 · 3 H2O 1.4 30
NaHCO3 15 300
H3BO3 0.19 3.7
MnCl2  4 H2O 0.42 8.3
FeCl3  6 H2O 0.16 3.2
Na2-EDTA · 2 H2O 0.30 6.0
ZnCl2 3.3 mg/L 66 µg/L
CoCl2  6 H2O 1.4 mg/L 29 µg/L
Na2MoO4  2 H2O 7.3 mg/L 145 µg/L
CuCl2  2 H2O 0.012 mg/L 0.24 µg/L
pH-value 7.5 ± 0.1
The pH of the test medium had to be 7.5  0.1 and was adjusted prior to testing with the addition of 1 N NaOH and HCl.

Application
Static with application of the test item at test start. At the start of the exposure, 3 uniform, healthy plants (colonies of 4 fronds each), were introduced into each test vessel containing the test media. The initial frond number per test vessel was 12. The initial numbers of colonies and fronds were the same in each test vessel.

Temperature (Target)
24 ± 2 °C

Light regime (Target)
Continuous, fluorescent light, 6500 to 10000 lux on the surface of the test medium (difference of light intensity at any measured incubation place < 15 % from the mean value)

Placement of the test vessels
A randomised placement of the test vessels was carried out.

Type and frequency of measurements
The numbers of plants and fronds were determined at the start and the end of the exposure. The number of fronds was determined every 2 - 3 days from each replicate of the control and the test concentrations. Every frond that visibly projected beyond the edge of a parent frond was counted as a separate frond. Fronds that lost their pigmentation were not counted.
Observations of frond size, appearance, indication of necrosis, chlorosis or gibbosity, colony break-up or loss of buoyancy, of root length and appearance, as well as of change in colour and destruction of roots, were made on every determination day and at the end of the exposure.
After 7 days, the determination of dry weight was carried out from 3 replicates per test concentration and 6 control replicates. Colonies from each test vessel were collected, rinsed with deionised water and then dried at 60 °C to a constant weight. Any root fragments were included. The dry weight was expressed to an accuracy of
0.1 mg.
The dry weight of the starting biomass was determined based on a sample of fronds (same number of fronds as in the test vessels) taken from the same batch used to inoculate the test vessels.

Physico-chemical Parameters
The pH-values were measured in the freshly prepared solutions before distribution into the replicates. The pH-values of the aged solution were measured from pooled replicates per concentration and control. The temperature of the medium in a surrogate vessel held under the same conditions in the growth room was recorded daily. The light intensity was measured prior to the start of the exposure at positions which had the same distance from the light source as the Lemna fronds.
Reference substance (positive control):
yes
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.44 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: frond number growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
6.45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: frond number growth rate
Remarks on result:
other: CI: 5.09 - 7.88
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.44 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Frond number yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.42 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: frond number yield
Remarks on result:
other: CI: 1.98 - 2.97
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.09 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.99 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight growth rate
Remarks on result:
other: CI: 2.48-3.66
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.44 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.96 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight yield
Remarks on result:
other: CI: 1.71 - 2.23
Details on results:
The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits.
Results with reference substance (positive control):
The acute toxicity of 3,5-Dichlorophenol (SIGMA, batch number SZBD168XV, purity 99.4%, CAS RN 591-35-5) to the monocotyledonous aquatic plant Lemna gibba was determined over a period of 7 days from 2016-10-12 to 2016-10-19 according to OECD Guideline 221. The plants used in the reference test were taken from the same laboratory culture as was used to determine the effects of Basic Red 18.1 Chloride.

EC50-Values of the Reference Item based on the nominal concentrations [mg/L], (0-7 days)
Current Study Valid Range (average ± 3 x SD)
Growth rate inhibition (number of fronds)
ErC50 6.71 5.56 ± 2.83
95% confidence interval 5.42 – 8.00
Yield inhibition (number of fronds)
EyC50 4.50 4.59 ± 3.03
95% confidence interval 4.04 – 5.05
Growth rate inhibition (dry weight)
ErdwC50 6.76 5.50 ± 2.85
95% confidence interval 5.29 – 7.43
Yield inhibition (dry weight)
EydwC50 5.53 4.63 ± 2.47
95% confidence interval 4.56 – 6.50
SD = standard deviation

The observed responses to the reference item were within the valid range, confirming the normal sensitivity of the test system used in the study with the test item.
Reported statistics and error estimates:
Sample size for statistics
For the determination of NOEC, LOEC and EC-values, three replicates were included for the test concentrations and six replicates for the control.

NOEC, LOEC and statistical analysess
The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield.
The following statistical tests were conducted:

Shapiro-Wilk’s test on normal distribution was done with a significance level 0.01.
Levene’s test on variance homogeneity was done with a significance level 0.01.
Monotonicity of concentration/response was done by trend analysis by contrasts (significance level 0.05).
Multiple sequentially-rejective Welsh-t-test after Bonferroni was carried out with a significance level 0.05.
William’s multiple sequential t-test was carried out with a significance level 0.05.


EC-values and statistical analyses
EC10-, EC20- and EC50-values (0 - 7 d) of the growth rate and yield (frond number and dry weight) inhibition were calculated by sigmoidal dose-response regression. Calculations of the confidence intervals of EC10-, EC20- and EC50-values were carried out from the best fit values, the standard error and the t-distribution with the software GraphPad Prism.

Frond Numbers

Geometric mean measured test item concentration
[mg/L]

Repl.

No.

Frond numbers per study day

0 days*

2 days

5 days

7 days

10.8

1

12

21

21

21

2

12

17

17

17

3

12

18

18

18

Mean

12

19

19

19

 5.05

1

12

24

54

58

2

12

23

46

56

3

12

22

42

52

Mean

12

23

47

55

 2.39

1

12

23

45

61

2

12

25

51

60

3

12

21

40

54

Mean

12

23

45

58

 1.09

1

12

24

52

67

2

12

24

52

72

3

12

23

58

76

Mean

12

24

54

72

 0.440

1

12

25

63

99

2

12

21

63

101

3

12

23

64

97

Mean

12

23

63

99

Control

1

12

25

73

118

2

12

21

61

95

3

12

25

76

115

4

12

21

52

97

5

12

25

66

113

6

12

20

62

102

Mean

12

23

65

107

* = 4 colonies with 3 fronds each per replicate were inoculated at start of the exposure

Repl. No. = replicate number

Growth Rate and Yield Inhibition based on Frond number

Statistically significant differences of growth rates and yield compared to control values are marked (+) and non-significant differences are marked (-).

                                               

Geometric mean measured test item concentration
[mg/L]

Repl.

No.

Average growth rate

[d-1]

Inhibition of average growth rate
[%]

Yield


[fronds]

Inhibition of yield

[%]

Doubling time

[d]

10.8

1

 

0.0799

74

 

9

91

8.67

2

 

0.0498

84

 

5

95

13.9

3

 

0.0579

81

 

6

94

12.0

Mean

(+)

0.0630

80

(+)

7

93

11.5

 5.05

1

 

0.225

28

 

46

51

3.08

2

 

0.220

29

 

44

54

3.15

3

 

0.209

33

 

40

58

3.31

Mean

(+)

0.218

30

(+)

43

54

3.18

 2.39

1

 

0.232

26

 

49

48

2.98

2

 

0.230

26

 

48

49

3.01

3

 

0.215

31

 

42

56

3.23

Mean

(+)

0.226

28

(+)

46

51

3.07

 1.09

1

 

0.246

21

 

55

42

2.82

2

 

0.256

18

 

60

37

2.71

3

 

0.264

15

 

64

32

2.63

Mean

(+)

0.255

18

(+)

60

37

2.72

 0.440

1

 

0.301

3

 

87

8

2.30

2

 

0.304

2

 

89

6

2.28

3

 

0.299

4

 

85

10

2.32

Mean

(-)

0.301

3

(-)

87

8

2.30

Control

1

 

0.327

 

 

106

 

2.12

2

 

0.296

 

 

83

 

2.35

3

 

0.323

 

 

103

 

2.15

4

 

0.299

 

 

85

 

2.32

5

 

0.320

 

 

101

 

2.16

6

 

0.306

 

 

90

 

2.27

Mean

 

0.312

 

 

95

 

2.23

Repl. No. = replicate number

 

 


Specific Growth Rate and Yield Inhibition of Dry Weight after 7 d

Statistically significant differences of specific growth rates and yield to control values are marked (+) and non-significant differences are marked (-).

 

Geometric mean measured test item concentration
[mg/L]

Repl.

No.

Dry weight


[mg]

Specific dry weight

growth rate

[d-1]

Inhibition of specific dry weight growth rate
[%]

Yield of dry weight


[mg]

Inhibition of yield dry weight
 
[%]

10.8

1

0.7

 

-0.109

100

 

-0.8

100

2

0.5

 

-0.157

100

 

-1.0

100

3

1.3

 

-0.020

100

 

-0.2

100

Mean

0.8

(+)

-0.095

100

(+)

-0.7

100

 5.05

1

4.3

 

0.150

50

 

2.8

74

2

3.5

 

0.121

60

 

2.0

81

3

3.4

 

0.117

61

 

1.9

82

Mean

3.7

(+)

0.129

57

(+)

2.2

79

 2.39

1

6.3

 

0.205

32

 

4.8

56

2

6.5

 

0.209

30

 

5.0

54

3

5.0

 

0.172

43

 

3.5

68

Mean

5.9

(+)

0.195

35

(+)

4.4

59

 1.09

1

6.8

 

0.216

28

 

5.3

51

2

9.0

 

0.256

15

 

7.5

31

3

9.2

 

0.259

14

 

7.7

29

Mean

8.3

(-)

0.244

19

(+)

6.8

37

 0.440

1

11.8

 

0.295

2

 

10.3

5

2

12.6

 

0.304

-1

 

11.1

-3

3

12.8

 

0.306

-2

 

11.3

-5

Mean

12.4

(-)

0.302

-1

(-)

10.9

-1

Control

1

13.5

 

0.314

 

 

12.0

 

2

11.5

 

0.291

 

 

10.0

 

3

13.1

 

0.310

 

 

11.6

 

4

11.5

 

0.291

 

 

10.0

 

5

12.7

 

0.305

 

 

11.2

 

6

11.4

 

0.290

 

 

9.9

 

Mean

12.3

 

0.300

 

 

10.8

 

The initial biomass dry weight was 1.5 mg per replicate.

Repl. No. = replicate number

 


Colony Number (Plants) on Days 0 and 7

 

Geometric mean measured test item concentration
[mg/L]

Replicate

No.


Colony number

Day 0

Day 7

10.8

1

3

17

2

3

14

3

3

15

Mean

3

15

 5.05

1

3

12

2

3

13

3

3

10

Mean

3

12

 2.39

1

3

7

2

3

6

3

3

5

Mean

3

6

 1.09

1

3

6

2

3

6

3

3

7

Mean

3

6

 0.440

1

3

9

2

3

9

3

3

9

Mean

3

9

Control

1

3

10

2

3

9

3

3

9

4

3

8

5

3

10

6

3

10

Mean

3

9


Further Observations on Days 2, 5 and 7

Geometric mean measured test item concentration
[mg/L]

Observations on day

2

5

7

10.8

2.4 +
3.3 +
4.1 ++

2.1 +
2.4 ++
3.3 ++
4.1 +++

2.1 +
2.4 ++
3.2 ++
3.3 ++
4.1 +++

 5.05

3.3 +
4.1 +

2.3 +
2.4 +
3.3 +
4.1 +

2.3 +
2.4 +
3.2 ++
3.3 ++
4.1 ++

 2.39

3.3 +

3.3 +

3.3 ++

 1.09

3.3 +

3.3 +

3.3 +

 0.440

1

1

1

Control

1

1

1

Observations were made compared to the appearance of control colonies (plants) and test media

 

1      = no observedeffects

2.1   = chlorosis

2.3   = gibbosity

2.4   = discoloration of fronds

3.2   = loses of roots

3.3   = discoloration of roots

4.1   = break up of plants

+      = slight effects

++    = medium effects

+++  = strong effects

Validity criteria fulfilled:
yes
Conclusions:
ErC50 = 6.45 (5.09 – 7.88) mg/L based on frond number
ErC50 = 2.99 (2.48 – 3.66) mg/L based on dry weight
Executive summary:

Method

The effects of the test item on the growth of the monocotyledonous aquatic plant species Lemna gibba was determined according to the principles of OECD 221.

Lemna gibba was exposed to the test item for 7 days under static conditions. Based on a preliminary test, 5 nominal test item concentration levels were tested in a geometrical series with a dilution factor of 2: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L, corresponding to the geometric mean measured test item concentrations: 0.440 – 1.09 – 2.39 – 5.05 – 10.8 mg/L. Three replicates were investigated for each test concentration and six for the control. Frond numbers were assessed on days 0, 2, 5 and 7. Environmental parameters (light, pH and temperature) were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

The concentrations of the test item and the control were analysed via HPLC-DAD at the beginning and end of the exposure.

The measured concentrationsat the start and the end of the exposure were in the range of 98 to 106 % and < LOQ to 110 % of the nominal values, respectively. All effect values are given based on the geometric mean measured test item concentrations.

Results

NOEC-, LOEC-, EC-Values and 95 % Confidence Intervals after 7 Days of Exposure  (based on the geometric mean measured concentration of the test item [mg/L])

Frond number

Dry weight

Growth Rate Inhibition [mg/L]

NOEC

0.440

NOEC

1.09

LOEC

1.09

LOEC

2.39

ErC10

0.932 (0.440 – 1.65)

ErdwC10

0.786 (0.587 – 0.991)

ErC20

2.14 (1.36 – 2.98)

ErdwC20

1.10 (0.857 – 1.35)

ErC50

6.45 (5.09 – 7.88)

ErdwC50

2.99 (2.48 – 3.66)

Inhibition of Yield [mg/L]

NOEC

0.440

NOEC

0.440

LOEC

1.09

LOEC

1.09

EyC10

0.444 (0.440 – 0.604)

EydwC10

0.537 (0.440 – 0.654)

EyC20

0.678 (0.482 – 0.881)

EydwC20

0.742 (0.605 – 0.882)

EyC50

2.42 (1.98 – 2.97)

EydwC50

1.96 (1.71 – 2.23)

Description of key information

ErC50 =   6.45 (5.09 – 7.88) mg/l based on frond number

ErC50 = 2.99 (2.48 – 3.66) mg/l based on dry weight

Key value for chemical safety assessment

Additional information

No studies on "Toxicity to aquatic plants other than algae" are available on the Target Substance in itself. However a study conducted on a Similar Substance has been used for the assessment.

Similar Substance 02 differ from the Target one only on the salification. More information are reported in section 13.

The effects of the test item on the growth of the monocotyledonous aquatic plant species Lemna gibba was determined according to the principles of OECD 221.

Lemna gibba was exposed to the test item for 7 days under static conditions. Based on a preliminary test, 5 nominal test item concentration levels were tested in a geometrical series with a dilution factor of 2: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L, corresponding to the geometric mean measured test item concentrations: 0.440 – 1.09 – 2.39 – 5.05 – 10.8 mg/L. Three replicates were investigated for each test concentration and six for the control. Frond numbers were assessed on days 0, 2, 5 and 7. Environmental parameters (light, pH and temperature) were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

The concentrations of the test itemand the controlwere analysed via HPLC-DAD at the beginning and end of the exposure.

The measured concentrations at the start and the end of the exposure were in the range of 98 to 106 % and < LOQ to 110 % of the nominal values, respectively. All effect values are given based on the geometric mean measured test item concentrations. The EC50 value based on growth rate calculated in this test are:

ErC50 =   6.45 (5.09 – 7.88) mg/L based on frond number

ErC50 = 2.99 (2.48 – 3.66) mg/L based on dry weight