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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-10-30 to 2018-01-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
adopted 2011-07-28
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identity: TMEDA, termed ATMEDAHP in the report
Purity: 99.9%
Lot number: 17F-1069978
C content: 62.01% (calculated)
Appearance: Liquid / colourless to yellow
Analytical monitoring:
yes
Details on sampling:
Samples of test media were taken at test initiation (0 h) and termination (72 h) for the analysis of TMEDA concentrations, to confirm that nominal exposure concentrations were achieved and satisfactorily maintained during the test. Samples were stored deep frozen during the interval between collection and preparation for analysis.
Vehicle:
no
Details on test solutions:
A weighed quantity of TMEDA was dissolved in AAP algal growth medium to prepare a primary 1000 mg/L stock solution. The primary stock was serially diluted with AAP medium to prepare a range of test media with nominal concentrations of 0.954, 3.05, 9.77, 31.3 and 100 mg TMEDA/L. Unamended AAP medium served as the control treatment.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was inoculated from a sterile preculture in exponential growth phase established at the test facility from a culture originally obtained from MBM Sciencebridge GmbH, Goettingen, Germany.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
Standard duration.
Post exposure observation period:
None, not a standard test requirement.
Hardness:
Not reported.
Test temperature:
22.9 to 23.3 degrees C.
pH:
0h: 7.62 (control), 8.40, 8.46, 8.92, 8.99 and 9.31 in TMEDA treatments in ascending concentration order.
72h: 7.95 (control), 7.90, 7.89, 7.97, 8.18 and 8.25 in TMEDA treatments in ascending concentration order
Dissolved oxygen:
Not relevant.
Salinity:
Not relevant (freshwater test).
Conductivity:
Not reported.
Nominal and measured concentrations:
See Table 6.1.5-1 below.
Details on test conditions:
Test media were continuously shaken on an incubated oscillating platform to maintain algal cells in suspension beneath continual illumination (75.9 microEinsteins/square metre/sec) provided by fluorescent lighting.
Reference substance (positive control):
yes
Remarks:
The most recent reference test with potassium dichromate prior to this study with TMEDA was performed during July 2017.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
20.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 11.7 to 37.9 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.82 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.09 to 9.03 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.41 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 4.46 to 16.2 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.09 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 0.123 to 2.71 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.954 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Measured initial TMEDA concentrations ranged from 104% to 113% of nominal and concentrations in aged samples recovered at the end of the test ranged from 108% to 124% of nominals. The biological response was therefore evaluated with reference to nominal concentrations.
Results with reference substance (positive control):
In the most recent reference test performed prior to this study with TMEDA, the 72h ErC50 was 1.23 mg K2Cr2O7/L.
Reported statistics and error estimates:
See table of endpoints (effect concentrations) above.

Table 6.1.5 -1: Concentrations of TMEDA in a test of growth inhibition of P. subcapitata

Nominal TMEDA concentration (mg/L)

Measured TMEDA concentration in algal growth medium
(mg/L)

0 h (fresh)
[% of nominal]

72 h (aged)
[% of nominal]

0 (control)

< LOD

-

< LOD

-

0.954

1.08

[113]

1.13

[118]

3.05

3.40

[111]

3.32

[109]

9.77

11.0

[113]

10.6

[108]

31.3

35.2

[112]

38.7

[124]

100

104

[104]

112

[112]

LOD:     0.0270 mg TMEDA/L

LOQ:     0.0900 mg TMEDA/L

 

Table 6.1.5-2: Algal cell densities and inhibition of growth rate and cell yield of P. subcapitata following 72-h exposure to TMEDA under static conditions

Nominal TMEDA concn (mg/L)

Mean algal cell density
(cells/mL × 10 000)

Inhibition of growth rate (%)

Inhibition of cell yield (%)

0 h

24 h

48 h

72 h

0-24 h

0-48 h

0-72 h

0-24 h

0-48 h

0-72 h

0

0.53

1.26

5.40

24.64

-

-

-

-

-

-

0.954

0.53

1.23

4.78

22.88

1.4

4.7

1.6

4.1

12.7

7.3

3.05

0.53

1.09

3.70

17.96

18.7

16.3

8.9*

23.3

34.9

27.7*

9.77

0.53

0.93

3.12

14.31

40.0

25.5

14.8*

45.2

46.8

42.8*

31.3

0.53

0.45

0.84

2.42

136.3

88.6

71.4*

111.0

93.6

92.2*

100

0.53

0.20

0.37

0.89

237.3

122.7

91.3*

145.2

103.3

98.5*

*             Statistically significant difference from the mean control

 

Table 6.1.5‑3: Analysis of control replicate growth data and derivation of coefficients of variation (CV) for test validity assessment

Control

Daily growth rate (d‑1)

mean

 

mean, section-by-section

0-24 h

0-48 h

0-72 h

0-72 h St Dev

0-72 h CV (%)

0-24 h

24-48 h

48-72 h

rep. 1

0.90240

1.22267

1.32158

0.36321

27

0.90240

1.54294

1.51941

rep. 2

0.89228

1.13580

1.30299

0.37837

29

0.89228

1.37933

1.63737

rep. 3

0.82774

1.20886

1.27498

0.39796

31

0.82774

1.58999

1.40722

rep. 4

1.09861

1.19692

1.32566

0.24369

18

1.09861

1.29523

1.58313

rep. 5

0.54430

0.96837

1.17083

0.55028

47

0.54430

1.39245

1.57575

rep. 6

0.84157

1.16704

1.24700

0.35371

28

0.84157

1.49252

1.40691

Mean

0.85115

1.14994

1.27384

-

30

0.85115

1.44874

1.52163

Std Dev

0.17908

0.09426

0.05856

-

-

0.17908

0.11162

0.09629

CV (%)

21.0

8.2

4.6

-

-

21.0

7.7

6.3

 

Validity criteria fulfilled:
yes
Remarks:
See 'overall remarks', above.
Conclusions:
The effects of TMEDA on the growth of the unicellular freshwater alga Pseudokirchneriella subcapitata were determined in a 72-hour static test performed according to OECD 201 (2011) and in compliance with GLP. The 72-h ErC50 causing a 50% reduction in specific growth rate compared to the unamended control was 20.5 mg TMEDA/L (95% CL: 11.7 to 37.9 mg/L). The ErC10 was 4.82 mg/L and the corresponding NOErC was 0.954 mg/L.
Executive summary:

Cells of the unicellular freshwater alga Pseudokirchneriella subcapitata were exposed for 72 h to TMEDA dissolved in AAP algal growth medium at nominal concentrations of 0 (control), 0.954, 3.05, 9.77, 31.3 and 100 mg/L. There were six replicate test vessels for the control group and three for each test substance treatment, each a 100 mL Erlenmeyer flask containing ca 50 mL medium initially inoculated with 5300 cells/mL. Test vessels were continually shaken to maintain cells in suspension and incubated under continuous illumination. Samples were taken from each flask after 24, 48 and 72 h to measure photosynthetic pigment fluorescence. Fluorescence measurements were converted to algal cell densities using a calibration established against direct cell counts made with a microscope and Neubauer chamber. Samples of test media were taken at test initiation and termination and analysed for TMEDA to confirm nominal exposure concentrations. Samples of media were taken from each treatment at 72 h and examined microscopically for evidence of morphological changes in the algal cells. A separate test was performed to determine the sensitivity of the stock culture to the reference toxicant potassium dichromate.

Analysis confirmed that exposure concentrations were achieved and maintained within 20% of nominals and biological effects were therefore evaluated on the basis of nominal exposure concentrations. Algal growth in the control replicates met the validity criteria of the guideline and the response to the reference toxicant was within the normal range; the observed response to TMEDA may therefore be considered reliable.

Statistically significant reductions in growth rate and cell yield occurred at concentrations >/= 3.05 mg TMEDA/L. No morphological abnormalities were observed in algal cells exposed to TMEDA and no detritus of ruptured cells was seen in any of the test media at 72 h. The 72 h ErC50 based on reduction of growth rate relative to the untreated control was 20.5 mg TMEDA/L (95% CL: 11.7 to 37.9 mg/L). The ErC10 was 4.82 mg/L and the corresponding NOErC was 0.954 mg/L.

Description of key information

The effects of TMEDA on the growth of the unicellular freshwater alga Pseudokirchneriella subcapitata were determined in a 72-hour static test performed according to OECD 201 (2011) and in compliance with GLP.  The 72-h ErC50 causing a 50% reduction in specific growth rate compared to the untreated control was 20.5 mg TMEDA/L (95% CL: 11.7 to 37.9 mg/L).  The ErC10 was 4.82 mg/L and the corresponding NOErC was 0.954 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
20.5 mg/L
EC10 or NOEC for freshwater algae:
4.82 mg/L

Additional information