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Ecotoxicological information

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:
2000-11-22 to 2001-05-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1984
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Remarks:
High Performance Liquid Chromatography (HPLC)
Details on sampling:
At the start of exposure (0 hr), 2.0 mL of the test solution was collected from each of the three test vessels in each test group and mixed, and the sample was used as an analysis sample. At the end (72 hr), 2.0 mL of the test solution was collected from each of the three test vessels in each test group and mixed, and the supernatant after centrifugation (3000 rpm, 10 minutes) of the algae was used as the analysis sample. An equal amount of acetonitrile was added to 0.75 mL of each analysis sample, mixed, and then analyzed by HPLC. The test substance concentration of each test solution was quantified from the ratio with the peak area of the standard solution.
Vehicle:
yes
Remarks:
0.01% (w/w): mixture of 2-methoxyethanol and HCO-60 in a ratio of 1 : 3 (w/w),
Details on test solutions:
92 μL of the test substance, which is equivalent to 100 mg, was collected, dissolved in 100 mg of 2-methoxyethanol, and then 300 mg of HCO-40 was added and mixed. This was diluted with a medium and the volume was adjusted to 100 mL to prepare a test substance solution having a test substance concentration of 1000 mg/L. At the same time, a solvent agent solution of 4000 mg/L (2-methoxyethanol 1000 mg/L, HCO-40 3000 mg/L) containing no test substance was prepared.
Based on these solutions, est item concentration groups were prepared to contain a constant vehicle concentration of 0.01 % by weight.
The control group used only the medium, and the solvent agent control group contained only the solvent agent (solvent agent concentration: 100 mg/L). The state (appearance) of the test solution at the time of preparation was colourless and transparent in all test groups.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test species:
1) Japanese name: Muremikazukimo (unicellular green algae)
2) Scientific name: Selenastrum capricornutum
3) Source: American Type Culture Collection
4) Obtained strain number: ATCC22662 Stock
5) Date of acquisition: June 20, 1996
6) Management after acquisition Aseptic subculture using Gorham medium
7) Sensitivity: 72 hours 50% algae growth inhibition concentration (EbC50) = 0.485 mg/L
with reference substance (potassium dichromate, technical grade reagent)
(This value is in the range of EbC50 value 0.285 to 0.529 mg/L, n = 7 since November 1996)
8) Pre-culture period: March 23, 2001-March 26, 2001; During this time, the algae grew logarithmically.
(Environmental conditions are the same as the test)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.181 mM (Ca/Mg) according to OECD medium
Test temperature:
22.4 - 23.8 °C
pH:
t=0: pH7.9

t= 72 h:
Controls and all but the two highest concentrations pH 10.5;
11.6 mg/L: pH 10.2;
25 mg/L: pH 8.2

Dissolved oxygen:
n.a.
Salinity:
n.a.
Conductivity:
No data
Nominal and measured concentrations:
Nominal test item concentration: 0.250, 0.540, 1.16, 2.50, 5.39, 11.6, 25.0 mg/L (common ratio: about 2.2)
Geometric mean measured test item concentrations:
0.124, 0.259, 0.574, 1.209, 2.613, 5.665, 12.552 mg/L
Details on test conditions:
I. Test conditions

1) Exposure method : Static, shaking intensity (100 rpm)
2) Exposure period : 72 hours
3) Test water volume : 100 mL (OECD medium)/vessel
4) Number of replicates : 3 test vessels/experimental design
5) Initial cell density : 1x10^4 cells/mL
6) Test water temperature : 23 +/- 2 °C
7) Lighting : Continuous lighting at 4000 lux
(within ± 20% fluctuation, near flask liquid level)

II. Dilution water

The medium specified in the OECD Chemical Test Guideline (OECD medium) was used for both preculture and testing.

III. Test container and algae culture test equipment, etc.

1) Test container : 500 mL Erlenmeyer flask with stopper
2) Algae culture test device : Ito Seisakusho AGP-150RL type
3) Optical microscope : Nikon ECLIPSE TE300 type
4) Particle counting device : Sysmex CDA-500
5) Electrolyte solution for particle counting device: Sysmex Xel-Há
6) pH meter : Desktop pH meter manufactured by Toa Denpa Kogyo HM-40V type No. 2
7) Thermometer: Tasco Japan TNA-120 type
8) Illuminometer: Topcon IM-2D type

IV. Test concentration design
Based on the results of the preliminary test (1 test vessel each) shown in the table below, the test concentration for the main test was determined as follows.
Test groups (three replicates, each):
control group, solvent control group, 0.250, 0.540, 1.16, 2.50, 5.39, 11.6, 25.0 mg/L (common ratio: about 2.2)

V. Observations and measurements
The cell concentration was measured by collecting 1.0 mL of the test solution from each test container, mixing it with 9.0 mL of the electrolytic solution for the particle counting device (cell pack), and then measuring it with the particle counting device (CDA-500). For the algae in the test solution, the colour tone was observed with the naked eye 0.24 and 48 hours after the start of exposure, and the colour tone was observed, and the cell morphology was observed under a microscope at the end of the exposure. The pH of the test solution in each test group was measured for one single vessel prepared separately from the three vessels in each test group at the start of exposure, and for one of the three vessels (No. 1) at the end of exposure (72 hr). During the exposure period, the temperature and illuminance in the incubator were measured at least once a day.

VI. Evaluations
Yield based inhibition was calculated as follows:
Growth inhibition (IA) by comparison of areas under the growth curve (Area-based method):
Area (A) calculation: A = [(N_1 - N_0)/2]*t_1 + [(N_1 + N_2 - 2*N_0)/2]*(t_2 - t_1) + ... + [(N_n-1 + N_n - 2*N_0)/2]*(t_n - t_n-1)
where N_0: Nominal cell concentration at the initiation of exposure (cells/mL)
N_i: Measured cell concentration at t_i (cells/mL)
N_n: Measured cell concentration at to (cells/mL)
t_1: Time of first measurement of cell concentration after initiation of exposure
t_n: Time of nth measurement of cell concentration after initiation of exposure
Calculation of inhibition based on area under the growth curve [%]:
I_A = [(A_c - A_t)/A_c]*100
where A_c: Area under the growth curve for solvent control; A_t: Area under the growth curve for the treatment group.

Growth rate based inhibition: Calculated according to paragraphs 48 through 51 of OECD TG 201 (2006).
Reference substance (positive control):
yes
Remarks:
Potassium dichromate, special grade reagent
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
6.09 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 5.92 - 6.26
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 8.71 - 9.52
Duration:
72 h
Dose descriptor:
EC5
Effect conc.:
5.43 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 5.28 - 5.59
Key result
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
4.97 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 4.84 - 5.12
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
8.44 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 8.14 - 8.75
Details on results:
All results given above are from the statistical re-evaluation performed in 2021. The original evaluation was performed based on initially measured values, and for the regulatory relevant endpoint growth rate for the exposure range between 24 and 72 h (instead of 0 - 72 h). Further, details on dose-response modelling were insufficient, and no confidence intervals were given.
Because the solvent control did not fulfil validity criteria according to OECD TG 201 (2006) for the last exposure interval (growth was not fully logarithmic any more between 48 and 72 h, see below for details), EC50 and EC10 were also derived for the interval 0-48 hours. Corresponding results demonstrate that there is no relevant underestimation of toxicity based on the effect values calculated for 0-72 hours.

For the endpoint reproduction rate, also NOECs were newly derived:
Exposure interval 0-72 h (normality test passed, variance homogeneity test passed, linear trend confirmed):
NOEC (0-72 h; growth rate) = 0.574 mg/L (Willilams test, a= 0.05)
LOEC (0-72 h; growth rate) = 1.279 mg/L (corresp. to 4.6% inhibition).

Exposure interval 0-48 h (normality test passed, variance homogeneity test failed)
NOEC (0-48 h; growth rate) = 2.61 mg/L (multiple sequentially-rejective Welsh t-test after Bonferroni-Holm, a= 0.05)
LOEC (0-48 h; growth rate) = 5.66 mg/L (corresp. to 20% inhibition).
Compared to the interval 0-72 h a significantly higher NOEC is obtained for the interval 0-48 h. This is mostly due to the fact that due to the variance heterogeneity a less powerful statistical test had to be performed.

Inhibition base on yield was not reevaluated. Originally reported values determined according to the area based method (based on measured initial concentrations) are the following:
EbC50 (0-72 h; yield) = 9.19 mg/L (no confidence interval)
NOEC (0-72 h; yield) = 3.69 mg/L
Results with reference substance (positive control):
The 72-hour growth inhibition concentration (EbC50) of the reference substance (potassium dichromate, special grade reagent) was 0.485 mg/L.
Reported statistics and error estimates:
Statistical evaluation was performed relative to the solvent control and based on geometric mean measured concentrations. ToxRatStd© v3.3.0 was used for all evaluations. Further details are given in the attached illustration.

Nominal and measured concentrations:






































































Nominal Measured Concentration (mg/L)
Concentration
(mg/L)
0 Hour72 HoursGeometric mean measured
Control<0. 003<0. 003 
Solvent  control<0. 003<0. 003 
0.2500.1700.0910.124
0.5400.3490.1920.259
1.1600.7990.4120.574
2.5001.6400.8911.209
5.3903.6901.8502.613
11.6008.2703.8805.665
25.00018.6008.47012.552

 


Detailed data on single replicate growth:




























































































































































































































































































































































































Nominal concentration
(mg/L)
Vessel Cell  Densities  (cells/mL) 
  0244872
 11000054900247600573800
Control21000050200208600466800
 31000046700214600516800
 Average1000050600223600519100
 SD041002100053500
 11000067100301600644800
Solvent. conc.21000068600324600585800
 31000065200322600592800
 Average1000067000316300607800
 SD017001270032200
 11000063400278600517800
0.25021000063200288600597800
 31000044300183600400800
 Average1000057000250300505500
 SD0110005800099100
 11000067800327600594800
0.54021000063300307600551800
 31000048500198600436800
 Average1000059900277900527866
 SD0101006940081700
 11000065100313600570800
1.16021000054800241600486800
 31000060700295600535800
 Average1000060200283600531100
 SD052003750042200
 11000054400220600451800
2.50021000066000307600537800
 31000063800298600524800
 Average1000061400275600504800
 SD062004780046400
 11000057500270600518800
5.39021000056200262600505800
 31000053900261600489800
 Average1000055900264900504800
 SD01800490014500
 11000041400158600410800
11.60021000042100156600403800
 31000042900159600402800
 Average1000042100158300405800
 SD080015004400
 110000158001780020200
25.000210000152001740018000
 310000164001710017000
 Average10000158001740018400
 SD06004001600

 


Validity criteria according to OECD TG 201 (2006)











































































































































































































































































































Solvent control, 0-72 h      
        
 Growth rate/dGrowth rate/dGrowth rate/dGrowth rate/dGrowth rate/dGrowth rate/d
 0 to 24 h24 to 48 h48 to 72 h0 to 72 h   
     SDCV 
Control Number      
11.903598951.502917590.759838561.388785030.5803590641.7889773 
21.925707441.554301120.590384781.356797780.6892222650.7977146 
31.874874381.59895370.608443911.360757330.6659689748.9410532 
        
MW1.901393591.552057470.65288908    
CV [%]0.013405010.030963640.1425358    
   Mean1.36878005   
   SD av.sp.gr.0.01743758Mean CV47.1759151limit:  <= 35%
  %CV av.sp.gr.1.27395028limit <= 7%  
        
        
Solvent control, 0-48 h      
        
 Growth rate/dGrowth rate/dGrowth rate/dGrowth rate/dGrowth rate/dGrowth rate/d
 0 to 24 h24 to 48 h48 to 72 h0 to 48 h   
     SDCV 
Control Number      
11.903598951.502917590.759838561.135505510.2833245124.9513985 
21.925707441.554301120.590384781.160002850.2626239322.6399381 
31.874874381.59895370.608443911.157942690.1951053816.8493126 
        
   Mean1.15115035   
   SD av.sp.gr.0.01358793Mean CV21.4802164limit:  <= 35%
  %CV av.sp.gr.1.18037826limit <= 7%  
Validity criteria fulfilled:
yes
Remarks:
Please refer also to section "overall remarks"
Conclusions:
In the reliable and adequate key study (MOE Japan, 2001) on fresh water green alga growth inhibition performed compliant with GLP according to OECD TG 201 (1984), the following results were obtained (based on geometric mean measured concentrations; in relation to solvent control):
ErC05 (0-72h) : 5.43 mg/L (95% confidence limit: 5.28 - 5.59)
ErC10 (0-72h) : 6.09 mg/L (95% confidence limit: 5.92 - 6.26)
ErC50 (0-72h) : 9.11 mg/L (95% confidence limit: 8.71 - 9.52)
NOEC (0-72 h; growth rate) = 0.574 mg/L (Willilams test, a= 0.05)
LOEC (0-72 h; growth rate) = 1.279 mg/L (corresp. to 4.6% inhibition).
Executive summary:

The reliable and adequate key study (MOE Japan, 2001) on fresh water green alga growth inhibition was performed compliant with GLP according to OECD TG 201 (1984). Selenastrum capricornutum was tested under static exposure conditions for 72 hours exposure. A control, solvent control and 7 nominal test item concentrations (0.250, 0.540, 1.16, 2.50, 5.39, 11.6, 25.0 mg/L) were set up with 3 replicates, each. All test item concentrations were analytically verified at the beginning and the end (72 h) of the test. Measured concentrations were between 65% and 74% of nominal after 0h and between 33 and 36% of nominal after 72 h. Water parameters were measured at the start and end of the test, temperature and illuminance in the incubator were measured at least once a day and found to be within requirements of the guideline. The pH varied from 7.9 at the start of exposure and 10.2 - 10.5 except for the highest test item concentration (8.2 - associated with very poor growth). Due to measured values below 80% of nominal, (no)effect values were derived based on geometric mean measured concentrations.
The following results were obtained with regard to the endpoint growth rate:


ErC05 (0-72h) : 5.43 mg/L (95% confidence limit: 5.28 - 5.59)
ErC10 (0-72h) : 6.09 mg/L (95% confidence limit: 5.92 - 6.26)
ErC50 (0-72h) : 9.11 mg/L (95% confidence limit: 8.71 - 9.52)
NOEC (0-72 h; growth rate) = 0.574 mg/L (Willilams test, a= 0.05)
LOEC (0-72 h; growth rate) = 1.279 mg/L (corresp. to 4.6% inhibition).

Description of key information

In the reliable and adequate key study (MOE Japan, 2001) on fresh water green alga growth inhibition performed compliant with GLP according to OECD TG 201 (1984), the following results were obtained (based on geometric mean measured concentrations; in relation to solvent control):
ErC05 (0-72h) : 5.43 mg/L (95% confidence limit: 5.28 - 5.59)
ErC10 (0-72h) : 6.09 mg/L (95% confidence limit: 5.92 - 6.26)
ErC50 (0-72h) : 9.11 mg/L (95% confidence limit: 8.71 - 9.52)
NOEC (0-72 h; growth rate) = 0.574 mg/L (Willilams test, a= 0.05)
LOEC (0-72 h; growth rate) = 1.279 mg/L (corresp. to 4.6% inhibition).

Key value for chemical safety assessment

EC50 for freshwater algae:
9.11 mg/L
EC10 or NOEC for freshwater algae:
6.09 mg/L

Additional information

The reliable and adequate key study (MOE Japan, 2001) on fresh water green alga growth inhibition was performed compliant with GLP according to OECD TG 201 (1984). Selenastrum capricornutum was tested under static exposure conditions for 72 hours exposure. A control, solvent control and 7 nominal test item concentrations (0.250, 0.540, 1.16, 2.50, 5.39, 11.6, 25.0 mg/L) were set up with 3 replicates, each. All test item concentrations were analytically verified at the beginning and the end (72 h) of the test. Measured concentrations were between 65% and 74% of nominal after 0h and between 33 and 36% of nominal after 72 h. Water parameters were measured at the start and end of the test, temperature and illuminance in the incubator were measured at least once a day and found to be within requirements of the guideline. The pH varied from 7.9 at the start of exposure and 10.2 - 10.5 except for the highest test item concentration (8.2 - associated with very poor growth). Due to measured values below 80% of nominal, (no)effect values were derived based on geometric mean measured concentrations.
The following results were obtained with regard to the endpoint growth rate:


ErC05 (0-72h) : 5.43 mg/L (95% confidence limit: 5.28 - 5.59)
ErC10 (0-72h) : 6.09 mg/L (95% confidence limit: 5.92 - 6.26)
ErC50 (0-72h) : 9.11 mg/L (95% confidence limit: 8.71 - 9.52)
NOEC (0-72 h; growth rate) = 0.574 mg/L (Willilams test, a= 0.05)
LOEC (0-72 h; growth rate) = 1.279 mg/L (corresp. to 4.6% inhibition).


No relevant supporting data are available.