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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:
2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Experimental study performed according to the OECD 201 guideline and under GLP conditions
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
N/A
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Please, see TMI
Analytical monitoring:
yes
Details on sampling:
The analytical samples were sampled at the beginning of the test in the test solutions preparations and at the end of the test in the content of the vessels pooled by replicate.
The following protocol was applied:
- Minimum of 1 mL of sample was collected and pipetted into a 2 mL micro-centrifugation tube;
- For control sample, LS/16, LS8, LS4 samples, 1 mL was collected with a syringe and was filtered (Filter Macherey Nagel RC-45/13 ref 729237) into a 1.5 mL glass amber vial;
- For LS/2 and LS samples, 100 µL was diluted with 900 µL of algae medium before filtration of 1 mL.
Details on test solutions:
Test item was supplied by the sponsor as a saturated solution. Aqueous phase of this solution was used as Limit of Solubility solution (LS). As this saturated solution was prepared with deionised water, stock solutions of media were added at LS solution as below:
Test item solution (LS) = 500 mL
Media stock solution (SM1) = 5 mL
Media stock solution (SM2) = 0.5 mL
Media stock solution (SM3) = 0.5 mL
Media stock solution (SM4) = 0.5 mL
Final volume (F) = 506.5 mL
Dilution factor (F / LS) = 1.013 mL
Dilution factor (1.013) was negligible to performed the test at LS concentration.
The test solution was then prepared by dilution of test item solution
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The Pseudokirchneriella subcapitata stock culture was maintained at 4°C and regularly transferred to fresh medium at the laboratory. The algal stock culture was provided by the CCAP (Culture Collection of Algae and Protozoa), reference CCAP 278/4. A culture was prepared from the stock culture and maintained in an exponential growth by successive transfers to fresh medium used in the test (OECD medium) every 3 to 4 days.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
None
Hardness:
Not specified
Test temperature:
22.4 to 23.0 °C (mean: 22.7°C)
pH:
7.6 - 8.0
Dissolved oxygen:
Not specified
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
Nominal: 0; LS/16; LS/8; LS/4; LS/2 and LS
Measured (T0): 0; 2.03, 3.98, 8.00, 16.37 and 32.36 mg/L
Measured (72h): 0; 2.01, 3.99, 7.99, 16.13 and 32.28 mg/L
Details on test conditions:
Based on the results of the no-GLP range-finding test, the test solutions were prepared in OECD medium at the definitive nominal concentrations of LS/16; LS/8; LS/4; LS/2 and LS. A control was performed at the same time with test media only.
Every flask was inoculated with the initial cell concentration of 5*10^3 cells.mL-1 of Pseudokirchneriella subcapitata as recommended in the guideline OECD 201.

Starting from a culture of 3.55E+06 cells per millilitres, 705 µL were inoculated in 500 mL of OECD medium for the control (C02) and 352.5 µL were inoculated in 250 mL of OECD medium for the tested concentrations (C03 to C07), to reach a concentration of about 5000 cells per milliliters. The preparations were divided in 6 replicates for the control and 3 replicates for the tested concentrations consisting in about 80 mL per replicates.
The flasks consisted of glass Erlenmeyer flasks of 250 mL capacity, closed with air-permeable stoppers made of polyether foam.
The test was performed in a thermostatic chamber maintained in the range of 21°C to 24°C controlled to ± 2°C under continuous illumination (with a light intensity between 4440 and 8880 lux). Algae were maintained suspended under continuous orbital stirring.
According to the study plan, cell biomass was measured every day in each flask using an electronic particle counter.
At the end of the test, after 72 hours of exposure, a microscopic observation was performed to verify a normal and healthy appearance of the inoculum culture and to observe any abnormal appearance of the algae.
pH was recorded at the beginning and the end of the test in all treatments.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 32.36 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
EC50 was above the solubility limit of the substance and could therefore not be determined
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
23.12 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
32.36 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Corresponding to the water solubility limit of the substance
Details on results:
See the section "any information on results incl. tables".
At the end of the test, after 72 hours of exposure, a microscopic observation was performed to verify a normal and healthy appearance of the inoculum culture and to observe any abnormal appearance of the algae.
Results with reference substance (positive control):
Potassium dichromate: EC-72h-50 (mg.L-1): 1.09
95% confidence limits (mg.L-1): 0.95 – 1.23

Table 1: Cells number on control flasks

 
 

Number of cells at 0 h

Measure at 24 h (cells / mL)

Measure at 48 h (cells / mL)

Measure at 72 h (cells / mL)

Control C02

5000

29608

206600

1474000

5000

27024

209100

1146000

5000

23256

172300

1211000

5000

18792

170100

1073000

5000

14384

159700

1277000

5000

29384

139000

1104000

Mean

5000

23741

176133

1214167

 

Table 2: Specific growth rates, growth rates and yields of control replicates

 

Specific growth rate 0 h – 24 h

Specific growth rate 24 h – 48 h

Specific growth rate 48 h – 72 h

Mean of specific rate per replicate

Standard deviation of specific rate

CV of specific rate

Growth rate (day-1) after 72h

CV growth 72h (%)

Yield (cells / mL) after 72h

CV yield 72h (%)

Control C02

1.779

1.943

1.965

1.895

0.102

5.370

1.895

2.1

1469000

12.2

1.687

2.046

1.701

1.812

0.203

11.220

1.812

1141000

1.537

2.003

1.950

1.830

0.255

13.931

1.830

1206000

1.324

2.203

1.842

1.790

0.442

24.687

1.790

1068000

1.057

2.407

2.079

1.848

0.704

38.122

1.848

1272000

1.771

1.554

2.072

1.799

0.260

14.465

1.799

1099000

Mean

1.526

2.026

1.935

1.829

0.328

17.966

1.829

1209167

 

Table 3: Cells number, growth rates and yields in the different replicates of the test item group

Rates

Number of cells at 0 h

Measure at 24 h (cells / mL)

Measure at 48 h (cells / mL)

Measure at 72 h (cells / mL)

Growth rate (day-1) after 72h

CV growth 72h (%)

Growth rate inhibition (%) compared to control after 72h

Yield (cells / mL) after 72h

CV yield 72h (%)

Yield inhibition (%) compared to control after 72h

mg test item/L after 72h

LS/16

(C03)

5000

24336

144200

1139000

1.809

3.4

1.06

1134000

19.7

6.22

5000

31776

191900

1578000

1.918

-4.88

1573000

-30.09

5000

14016

140500

1144000

1.811

0.98

1139000

5.80

Mean

5000

23376

158867

1287000

1.846

-0.95 (ns)

1282000

-6.02 (ns)

LS/8

(C04)

5000

29392

158300

1025000

1.774

2.8

2.98

1020000

16.0

15.64

5000

29504

179000

1335000

1.862

-1.83

1330000

-9.99

5000

29016

181600

1024000

1.774

3.00

1019000

15.73

Mean

5000

29304

172967

1128000

1.804

1.38 (ns)

1123000

7.13 (ns)

LS/4

(C05)

5000

18728

153400

1065000

1.787

1.9

2.28

1060000

10.4

12.34

5000

29456

182200

1289000

1.851

-1.20

1284000

-6.19

5000

32928

158000

1106000

1.800

1.60

1101000

8.95

Mean

5000

27037

164533

1153333

1.813

0.89 (ns)

1148333

5.03 (ns)

LS/2

(C06)

5000

31240

151800

560400

1.573

3.3

13.99

555400

16.0

54.07

5000

20328

148900

769900

1.679

8.20

764900

36.74

5000

13192

123300

651900

1.623

11.23

646900

46.50

Mean

5000

21587

141333

660733

1.625

11.14 (ns)

655733

45.77 (ns)

LS (C07)

5000

27952

146000

642600

1.619

1.0

11.49

637600

5.1

47.27

5000

29928

152400

704700

1.649

9.81

699700

42.13

5000

21640

128300

652200

1.624

11.22

647200

46.48

Mean

5000

26507

142233

666500

1.631

10.84 (ns)

661500

45.29 (ns)

ns: not statisticallysignificant compared to the control (p>0.05, Dunn test)

* statistically significant compared to the control (p<0.05, Dunn test)

 

Table 4: pH measurements

C02

C03

C04

C05

C06

C07

pH at 0 h

7.7

7.7

7.7

7.7

7.7

8.0

pH at 72 h

7.7

7.6

7.6

7.6

7.6

7.9

 

Table 5: Temperature and light intensity during the test

Time

Mean

Min

Max

Temperature (TMP_0073)

22.7

22.4

23.0

Light intensity (lux)

8085

Table 6: Validity criteria

Actual value

Reference value

Compliance

Growth multiplication factor in the control within the 72-hour test period

243

16

Y

Coefficient of variation of the average specific growth rates in the control during the whole test period

2.12

7

Y

Mean coefficient of variation for section-by-section specific growth rates (days 0-1. 1-2 and 2-3) in the control

17.97

35

Y

Table 7: Analytical results

ID

Concentrations

0h(mg.L-1)

72h(mg.L-1)

C02

0

0

0

C03

LS/16

2.03

2.01

C04

LS/8

3.98

3.99

C05

LS/4

8.00

7.99

C06

LS/2

16.37

16.13

C07

LS

32.36

32.28

Validity criteria fulfilled:
yes
Conclusions:
RDX was not acutely toxic to Pseudokirchneriella subcapitata when tested at the solubility limit of the compound. EC50 based on growth rate could not be determined because it was above the solubility limit of the substance, i.e. 32.36 mg/L.
The lowest-observed-effect concentration (LOEC) and no-observed-effect concentration (NOEC) were > 32.36 and 32.36 mg/L, respectively. The EC10 was 23.12 mg/L (95% confidence limits: 15.23 – 33.44 mg/L).
Executive summary:

The objective of this study was to assess the effects of test item Hexogen solution on the growth of Pseudokirchneriella subcapitata over a period of 72 hours according to OECD 201 Guideline. Pseudokirchneriella subcapitata is a freshwater green algae that is recommended by the OECD Guideline 201.

The test item is a saturated solution supplied by the sponsor. The test solutions were prepared in the test medium by dilution of saturated test item solution, and were expressed in limit of solubility (LS). The effects of Hexogen solution at the definitive nominal concentrations of 0; LS/16; LS/8; LS/4; LS/2 and LS to Pseudokirchneriella subcapitata were investigated under laboratory conditions.

The analytical samples were sampled at the beginning of the test and at the end of the test (72 hours) with the content of the vessels pooled and homogenized.

Since the deviation between the measured concentrations at T0 and the measured concentrations at 72h was within the range of ± 20 %, analysis of the results were based in the measured concentrations at T0.

The EC10, EC20, EC50 and NOEC/LOEC values are summarized in the table below:

 

Hexogen solution

NOEC

(growth rate and yield)

32.36 mg.L-1 

LOEC

(growth rate and yield)

>32.36 mg.L-1 

72h-ErC10

(95% confidence limits)

23.12 mg.L-1(95% confidence limits: 15.23 – 33.44 mg.L-1)

72h-ErC20

(95% confidence limits)

>32.36 mg.L-1 

72h-ErC50

(95% confidence limits)

>32.36 mg.L-1 

72h-EyC10

(95% confidence limits)

5.65 mg.L-1(95% confidence limits: 1.76 – 14.44 mg.L-1)

72h-EyC20

(95% confidence limits)

10.72 mg.L-1(95% confidence limits: 5.27 – 20.07 mg.L-1)

72h-EyC50

(95% confidence limits)

31.98 mg.L-1(95% confidence limits: 22.28 – 49.57 mg.L-1)

EC = Effect concentration

NOEC= No Observed Effect Concentration

LOEC = Lowest Observed Effect Concentration

Description of key information

For that endpoint, an experimental study was available to assess the effects of test item on the growth of Pseudokirchneriella subcapitata over a period of 72 hours according to OECD 201 Guideline and under GLP conditions.

Since the deviation between the measured concentrations at T0 and the measured concentrations at 72h was within the range of ± 20 %, analysis of the results were based in the measured concentrations at T0.

RDX was not acutely toxic to Pseudokirchneriella subcapitata when tested at the solubility limit of the compound. EC50 based on growth rate was above the solubility limit of the substance, i.e. 32.36 mg/L.

The lowest-observed-effect concentration (LOEC) and no-observed-effect concentration (NOEC) were > 32.36 and 32.36 mg/L, respectively. The EC10 was 23.12 mg/L (95% confidence limits: 15.23 – 33.44 mg/L).

All validity criteria were met, therefore this study was considered valid.

Key value for chemical safety assessment

Additional information

For that endpoint, quite similar results were found in the study from Burton et al. (publication, 1994) performed on RDX according to EPA TSCA Method 797.1060 (U.S. EPA, 1985 and 1986).

In this study, a maximum reduction of 38% in cell density occured after a 96-h exposure to the solubility limit of the substance, i.e. 36.7 mg/L ; thus, an EC50 could not be determined.

The lowest-observed-effect concentration (LOEC) and no-observed-effect concentration (NOEC) for the green alga Selenastrum capricornutum were 4.8 and 0.5 mg/L, respectively, when chronic endpoints were used to analyze the data. Nevertheless, the key parameter investigated was the inhibition of growth after 96 hours (i.e. 96h-EC50) based on yield rather than growth rate (logarithmic increase in biomass). Therefore this study does not provide an adequate coverage of the key parameter foreseen to be investigated. In addition, the mean coefficient of variation for section-by-section specific growth rates in the control cultures was above 35% meaning that the validity criteria were not all successfully met when compared with those of the test methods referred to in Article 13(3), i.e. EU C.3 and OECD TG 201. For these reasons, this study has been finally disregarded.