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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:
1998-12-01 to 1998-12-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study, non GLP, analytical monitoring, closed vessels
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
other: Official Journal of the European Communities, L383, Part C.3, Algal inhibition test
Version / remarks:
29 December 1992
Principles of method if other than guideline:
- Parameters analysed / observed: 4 replicates in control and 2 instead of 3 replicates in each dose group. 7 dose groups were tested
- Initial cell density at start: 0.1 x 10 E4 cells/ml instead of 5 x 10 E3 to 10E4 cells/ml for pseudokirchneriella subcapitata
- One blank vessel (without algal inoculum) was incubated concurrently for each control and test concentration sampling occassion. These vessels were used for blank particle counting at each time point and specific analysis at the end of the test period.
GLP compliance:
no
Specific details on test material used for the study:
Methacrylic acid (CAS: 79-41-4)
Purity: 99.87%; supplied by ICI Acrylics. Certificate of Analysis stated sample purity determined by GC.
Clear and colorless liquid, stored under refrigeration.
Analytical monitoring:
yes
Details on sampling:
At the start of the test, samples were taken of each test solution, using the excess remaining after filling the test vessels, and were analyzed by HPLC for test substance concentration. At the end of the test each blank solution was sampled and analyzed in the same manner.
Vehicle:
no
Details on test solutions:
Two stock solutions were prepared by direct addition of the test substance to 1000 ml of sterile culture medium to give final concentrations of 410 and 10000 mg/L. The three lowest concentrations were prepared by addition of aliquots of the nominal 410 mg/L solution to sterile medium, to a final volume of 1000 ml. The remaining test concentrations were prepared by addition of aliquots of the nominal 10000 mg/l solution to sterile medium, to a final volume of 1000 ml. Test solutions were all clear and colorless.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Selenastrum capricornutum Printz, strain ATCC 22662
Test organisms were from laboratory cultures maintained under axenic conditions. A culture in the exponential growth phase was used as inoculum for the test.
The culture was grown in the medium, and under the environmental conditions, describend for the test.
Culture medium
The culture medium used for the test, and for the maintenance of cultures of the alga used as inoculum for the test based on that described
by Miller et al , with an increased NaHCO3 concentration (equivalent to 114 mg/l) to serve as an extra carbon ( dioxide) source.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
standard test medium used
Test temperature:
24°C
pH:
see : Details on test conditions (table: Test solution pH measurements)
Nominal and measured concentrations:
Nominal = 0.86, 1.9, 4.1, 9.1, 20, 45 and 100 mg/L
Geometric mean measured = 0.85, 2.0, 3.7, 8.2, 19, 45, 100 mg/L (measurement performed in blank solutions after 24 h and 72 h without algae)
Details on test conditions:
Test vessels were borosilicate glass bottles of 50 ml nominal capacity, filled completely with airtight, teflon faced disc/crimp closures. Cultures were incubated at 24+/-2°C under continous "cool white" illumination with orbital shaking. Four replicates of the control and duplicates of the test substance were employed. At 24, 48 and 72 hours replicate vessels were sacrificed and counted.
Each Flask contained 100 ml of test solution.
Incubation: at 24 +/- 2 °C, "cool-white" illumination, orbital shaking at 160 rpm
Inoculum: Cell density 0.10 x 10 E4 cell/ml
pH was measured at the start of the test and at the end of the test the pH of two replicate test solutions (containing algae) from each control and test concentration was determined.
light intensity: 8270 lux (by cosine receptor)

CULTURE MEDIUM FOR Pseudokirchneriella subcapitata
To approximately 900 ml of distilled water 1 ml of solutions Al, A2, A3 and B were added.
Distilled water was autoklaved at 103 kPa for 15 minutes.evaporation of distilled water which is replaced by sterile addition).
7.67 ml of solution C (aseptic technique) were added.

Solution A1
NaNO3: 12.75 g
MgCl2.6H20: 6.082 g
CaCl2.2H20: 2.205 g
Distilled water to: 500 ml

Solution A2
MgSO4.7H2O: 7.35 g
Distilled water to: 500 ml

Solution A3
K2HPO4.3H20: 0.684 g
Distilled water to: 500 ml

Solution B (Micronutrients)
H3BO3: 0.093 g
MnCl2-4H20: 0.208 g
FeCl3.6H20: 0.080 g
Na2EDTA.2H20: 0.150 g
ZnCl2: 1.64 mg
CoCl2.6H20: 1.714 mg
Na2MoO4.2H2O 3.63 mg
CuCl2.2H2O 0.006 mg
Distilled water to 500 ml

Solution C
NaHCO3 7.5 g
Distilled water to 500 ml
(Filter sterilise into sterile vessel)

NB: All weights and volumes described are nominal values.

Test solution pH measurements
Mean measured concentration of methacrylic acid
(mg/l) pH
Day 0 Day 3
Replicate A Replicate B
< 0.031
(control) 9.27 9.69
0.85 8.12 9.21 9.39
2.0 8.18 9.38 9.11
3.7 8.21 9.04 9.04
8.2 8.12 8.76 8.61
19 7.99 7.48 7.39
45 7.37 7.04 6.98
105 6.39 6.18 6.11



Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
8.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass
Remarks on result:
other: calcutated based an conc in blank solution (without algae)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: calcutated based an conc in blank solution (without algae)
Remarks:
95 % confidence limits: 37 to 58 mg/l
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
20 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: calcutated based an conc in blank solution (without algae)
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
19 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: calcutated based an conc in blank solution (without algae)
Details on results:
Mean measured concentrations of the test substance ranged from 90 -105 % of nominal exposure values. The percentage change in the measured concentrations over the test period ranged from a measured loss of 17 % to a measured increase of 10 %. On the basis of this analysis mean measured test conc in the blank solutions were used for the calculation of the biological results.
The authors stated that the solutions in the exposure flasks gave variable results, probably due to adsorption or metabolism of the methacrylic acid, so that these data have not been used in the calculaion of effect concentrations.
Reported statistics and error estimates:
Recalculated statistic results of the validity criteria:
Criteria for mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures is ≤ 35%: Pass as, the mean coefficient of variation is 14.7.
- Criteria for coefficient of variation of average specific growth rates during the whole test period in replicate control cultures is ≤ 7% in tests with Pseudokirchneriella subcapitata or Desmodesmus subspicatus. For other less frequently tested species, the value is ≤ 10%;
Pass as, coefficient of variation of average specific growth rates is 5.11.

MEAN AEREAS UNDER THE GROWTH CURVE AND PERCENTAGES OF THE CONTROL; INDICATING STATISTICALLY SIGNIFICANT DIFFERENCES

Mean measured conc of methacrylic acid

(mg/l)

Mean area under growth curve (0-3 days)

%

control

< 0.031

(control)

23.2

-

0.85

19.5

84

2.0

18.6

80

3.7

17.1

74

8.2

19.4

84

19

13.7*

59

45

3.3*

14

105

0.2*-

1

* Significant difference from the control (P=0.05)

MEAN GROWTH RATES OVER THE TEST PERIOD AND PERCENTAGES OF

THE CONTROL, INDICATING STATISTICALLY SIGNIFICANT DIFFERENCES

 

Mean measured conc of methacrylic acid(mg/l)

Mean growth rate

(0 - 3days)

%

of control

<0.031

(Control)

1.935

-

0.85

1.840

95

2.0

1.841

95

3.7

1.778

92

8.2

1.855

96

19

1.657*

86

45

1.223•

63

105

0.124*

6

*Significant difference from the control (P=0.05)

Algal cell densities

Measured conc of

methacrylic acid

(mg/l)

Replicate

Algal cell density (x 10 E4 cell /ml)

 

 

Day 1

Day 2

Day 3

< 0.031

1

2

3

4

0.877

0.906

0.828

0.770

6.36

5.31

6.02

6.33

22.2

39.8

28.9

41.7

Mean

0.845

6.01

33.2

0.82

1

2

0.821

0.813

5.18

7.70

19.4

30.6

Mean

0.817

6.44

25.0

2.0

1

2

0.783

0.850

4.84

6.15

29.4

20.7

Mean

0.817

5.50

25.1

3.7

1

2

0.979

0.866

6.29

5.87

21.6

19.8

Mean

0.923

6.08

20.7

8.2

1

2

0.958

0.758

5.21

6.35

27.3

24.9

Mean

0.858

5.78

26.1

19

1

2

0.464

0.757

6.35

5.96

16.8

12.0

Mean

0.611

6.16

14.4

45

1

2

0.410

0.186

0.944

1.69

4.53

3.32

Mean

0.298

1.32

3.93

105

1

2

0.222

0.126

0.195

0.125

0.199

0.091

Mean

0.174

0.160

0.145

Inoculum (0 hour) cell density = 0.1 x 10E4 x cell/ml

Validity criteria fulfilled:
yes
Conclusions:
In a valid guideline study, the 72 hr EbC50 and ErC50 were 20 and 45 mg/L, respectively for MAA with Selenastrum capricornutum (new name: Pseudokirchnerella subcapitata). The NOEC for growth and biomass was 8.2 mg/L.
Executive summary:

In a valid acute alga growth inhibition test acc. OECD 201 and Official Journal of the European Communities, L383, Part C.3, Algal growth inhibition test, methacrylic acid was tested against Selenastrum capricornutum (new name: Pseudokirchnerella subcapitata) in 7 test concentrations in duplicate at nominal concentrations of 0.86, 1.9, 4.1, 9.1, 20, 45 and 100 mg/L (corresponding to geometric mean measured concentrations of 0.85, 2.0, 3.7, 8.2, 19, 45, 105 mg/L, measured concurrent blank solutions without algae) for 72 hours in closed vessel under static conditions. EbC50 (biomass) was 20 mg/l, ErC50 (growth rate) was 45 mg/L, NOEC for growth and biomass was 8.2 mg/L based on geometric mean measured concentrations. Substance loss over test period was up to 17 % to a measured increase of 10% in blank solution.

Description of key information

In a valid acute alga growth inhibition test acc. OECD 201 and Official Journal of the European Communities, L383, Part C.3, Algal growth inhibition test, methacrylic acid was tested against Selenastrum capricornutum (new name: Pseudokirchneriella subcapitata) in 7 test concentrations in duplicate at nominal concentrations of 0.86, 1.9, 4.1, 9.1, 20, 45 and 100 mg/L (corresponding to geometric mean measured concentrations of 0.85, 2.0, 3.7, 8.2, 19, 45, 105 mg/L, measured concurrent blank solutions without algae) for 72 hours in closed vessel under static conditions. EbC50 (biomass) was 20 mg/l, ErC50 (growth rate) was 45 mg/L, NOEC for growth and biomass was 8.2 mg/L based on geometric mean measured concentrations. Substance loss over test period was up to 17 % to a measured increase of 10 % in blank solution.

Key value for chemical safety assessment

EC50 for freshwater algae:
45 mg/L
EC10 or NOEC for freshwater algae:
8.2 mg/L

Additional information

In 1990 an alga growth inhibition test (MPA) was performed under GLP. In a preliminary test Methacrylic acid was tested at 0.10, 1.0, 10 and 100 mg/l. Effects were observed at the highest dose of 100 mg/l. Based ob these results a definitive study was performed at 6.3 to 1000 mg/l but was terminated due to severe effects after 48 hours the results were used as preliminary study and the definitive study was based on these results performed with 5 test concentrations of 0.38, 0.75, 1.5, 3.0 and 6.0 mg/l.

Several studies have been performed (Brixham a, b, c, d) to explain the low effect concentrations in which the changing of different parameters have been observed (open and closed vessels, non-pH-adjustment and pH-adjustement, exclusion of macro-and micronutients). It has been shown that exclusion of NaHCO3 would severely reduce the buffering capacity of the media which would allow a high pH-shift on addition of 6 mg/l methacrylic acid (pH lowered to 4.6).

The key study was performed in standard medium at pH 8.12 (0.85 mg/l and pH 7.37 (45 mg/l) at day 0 and pH 9.21 (0.85 mg/l) and pH 6.48 (45 mg/l) at day 3.

The MPA (1990) is considered to have been performed without NaHCO3 which results in a pH shift and according to this in low effect concentrations. Therefore the study was considered to be not reliable.

Additional supporting information is available of marine algae. In a test with S. costatum acc. ISO 10253 (Sverdrup 2001) EC50 (72h) was > 1260 mg/l, NOEC (72h) was 530 mg/l measured conc. (based on growth rate). 9 other marine algae have been tested (Sverdrup 2001) for 2 -5 days. All EC50 values were > 100 mg/l based on growth rate. However, these studies havn't been reported in sufficient details.

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