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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:
14 September 1994 - 5 January 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples of 3.5 ml (main test 1) or 1-2 ml (main test 2) were taken from the test solutions containing algae after 24, 48, 72 and 96 hours of exposure.
Vehicle:
yes
Details on test solutions:
Nominal concentrations were prepared by dissolving AHTN in 5 ml of dimethylformamide (DMF). This was mixed with Tween 80, followed by a subsequent dilution with aqueous test medium, to form a stock solution.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was run with the recommended species Pseudokirchneriella subcapitata (strain no. 61.81 SAG). These test organisms, formerly known as Selenastrum capricornutum were suppiled by “Pflanzenphysiologisches Institut der Universitât Göttingen, D-37073 Göttingen). The algae were grown in the laboratories of RCC under standardized conditions.
Three days prior to the test a pre-culture of algae was inoculated under the same conditions as in the test. From this pre-culture the algae cells were taken to prepare an algae suspension in test medium. Aliquots of this algae suspension (corresponding to a biomass of 10,000 cells per ml of nutrient solution) were mixed with the respective test concentrations in test medium.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
The hardness of the test medium corresponded to about 24 mg CaCO3/l.
Test temperature:
Room temperature
pH:
The pH for all test concentrations was 8.0 at the start of the test after equilibration with air and ranged from 9.0 to 10.1 at the end of the test. This slightly exceeds the tolerance of 1.5 pH units given by the guidelines. Because rapid growth and hence the large CO2 consumption, this could not be avoided, despite stirring at 500 rpm.
Dissolved oxygen:
Solution contibuously stirred at 500 rpm.
Nominal and measured concentrations:
See table 1 in "any other information on results incl. tables" section.
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 835 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Test B
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
404 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Geometric mean of tests A and B
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
816 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Geometric mean of tests A and B
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
625 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Geometric mean of tests A and B
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
276 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Geometric mean of tests A and B
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
605 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Geometric mean of tests A and B
Results with reference substance (positive control):
The EbC50 and ErC50 for Potassium Dichromate after 72 hours of exposure was found to be at 0.53 mg/l (0.20 to 0.75 mg/l), and 0.84 mg/l (0.60 to 1.03 mg/l), respectively. Therefore, the algal cultures used by RCC were demonstrated to be adequately sensitive.
Reported statistics and error estimates:
As far as possible, the EbC50 and ErC and there confidence limits were estimated using the Logit model. The NOEC and LOEC were determined by applying Dunnett’s test.
For the summary, geometric means were calculated for the NOEC, LOEC and EC50 values in test A and test B.

Potassium dichromate as reference compound showed after 72 hours of exposure an EbC50 of 0.53mg/l and ErC50 of 0.84mg/l. It means that the test is sufficient sensitive. The pH was initially 8 at the start of the experiment and increased to 9 -10 at the end.

Table 1. Nominal and measured AHTN concentrations in the samples

Nominal concentration of test article
(µg/l)
Time
hours
% of nomal found Mean measured concentration of test article
(µg/l)
% of nomal found Mean measured concentration of test article
(µg/l)
62.5 0 82.6 77.4
72 30.8 140
mean 56.7 35.4 108.7 67.9
125 0 80.7 81.4
72 60.2 142
mean 70.5 88.1 111.7 139.6
250 0 90.1 83.5
72 72.8 52.7
mean 81.5 203.8 68.1 170.3
500 0 89.7 84.4
72 85.3 67.8
mean 87.5 437.5 76.1 380.7
1000 0 84.7 90.4
72 74.7 76.6
mean 79.7 797 83.5 835.0

The results of the inhibition test and algal growth can be found in table 2.

Table 2 AHTN-related inhibition of algal growth after 72 hours of exposure

Concentration of test article (µg/l) % Inhibition
Nominal Mean measured Biomass (AUC)
(0-72 hours)
Growth rate (µ)
Test 1 Test 2 Test 1 Test 2 Test 1 Test 2
35.4 67.9 17.8 -4 3 1.6
88.1 139.6 25.4 -11.8 6 -3.1
203.8 170.3 17.4 -20.9 -3 -2.1
437.5 380.7 36.0 -12.4 8.4 0.5
797.0 835.0 81.6 54.0 33.1 16.4

AUC: Areau Under Curve

The effect concentrations for the biomass and algal growth is shown in table 3

Table 3 NOEC, LOEC and EC50 for biomass and growth rate

Biomass Test 1 Test 2
NOEC: 204 µg/l 381 µg/l
LOEC: 438 µg/l 835 µg/l
EbC50: 468 µg/l 801 µg/l (extrapolated value)
Growth rate (µ): Test 1 Test 2
NOEC: 438 µg/l 381 µg/l
LOEC: 797 µg/l 835 µg/l
ErC50: >797 µg/l >835 µg/I
Validity criteria fulfilled:
yes
Remarks:
See overall remarks.
Conclusions:
The toxicity of AHTN for Pseudokicrhneriella subcapitata was studied in two tests. The geometric mean for the 3d-NOEC for growth rate (µ) is 404 µg/l and ErC50 is above 835 µg/l. The geometric mean for the 3d-NOEC (biomass, AUC) is 276 µg/l and the 3d-EbC50 is 625 µg/l.
Executive summary:

The toxicity to algae was studied in a static test on Pseudokirchneriella subcapitata according to OECD TG 201. Nominal concentrations ranged from 0.0625 to 1.0 mg/l (step size 2). Concentrations were measured at the start and end of the test by HPLC analysis. Maintenance of the concentrations proved to be difficult and in some cases pH-values rose to above 10 indicating an insufficient buffering capacity of the medium. The loss of test material in the algal test was studied for a related polycyclic musk and it was shown that sorption to algal cells was the most relevant ‘sink’, whereas sorption to glass might play a role as well as volatilisation. The results were expressed as the mean concentration over the exposure time. The test was carried out twice (test A and B) due to the sharp concentration decrease in the first study with the goal to confirm the observed toxicity levels in a second study. Growth rate (μ) was the less sensitive parameter. In test (B) the growth rate reduction at 0.835 mg/l was 16% (LOECr) with a NOECr of 0.374 mg/l. In test (A), the NOECr for effects on growth rate was slightly higher, 0.438 mg/l, whereas the inhibition at 0.797 mg/l (LOECr) was 33%. The geometric mean NOECr for both tests was 0.40 mg/l. The 72-h ErC50 was > 0.797 and > 0.835 in test (A) and (B) respectively. Growth measured as Area Under the Curve was not significantly inhibited in concentrations up to and including (A) 0.204 and (B) 0.374 mg/l. In the next higher concentration, the LOECb, (A) 0.438 and (B) 0.835 mg/l, the biomass production was inhibited by (A) 36% and (B) 54%.

The geometric mean for the 3d-NOEC for growth rate (µ) is 404 µg/l and ErC50 is above 835 µg/l. The geometric meand for the 3d-NOEC (biomass, AUC) is 276 µg/l and the 3d-EbC50 is 625 µg/l.

Description of key information

The toxicity for the freshwater alga (Pseudokirchneriella subcapitata) was studied according to OECD TG 201 and under GLP (Rel. 1). The 3d-NOErC is 404 µg/L and ErC50 is above 835 µg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.835 mg/L
EC10 or NOEC for freshwater algae:
0.404 mg/L

Additional information

The toxicity of the test item for Pseudokirchneriella subcapitata was studied in two tests. The results were in line with each other. As the endpoints were identical, the results are presented as the geometric means.

The geometric mean was calculaterd for the 3d-NOEC for growth rate (µ): 0.404 mg/L (A: 0.438 and B: 0.374 mg/L). The ErC50 was above 0.797 (A) and above 0.835 mg/L (B).

The geometric mean for the 3d-NOEC (biomass, AUC) is 0.276 mg/L (A: 0.204 and B: 0.374 mg/L) and the 3d-EbC50 is 0.625 mg/L (A: 0.468 and B: circa 0.835 mg/L).