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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 - 24 Sep 2015
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Germany

Test material

Constituent 1
Details on test material:
- Name of test material: reaction mass of potassium sodium (2R,3R)-2-hydroxy-3-(phosphonatooxy)butanedioate and potassium sodium (2S,3S)-2-hydroxy-3-(phosphonatooxy)butanedioate
- Physical state: white to slightly yellow powder of crystalline appearance
- Lot/batch No.: 010-2065
- Expiration date of the lot/batch: 16 Sep 2019
- Chemical stability: insensitive to exposure to light, considered to be stable in alkaline and neutral solutions (6 < pH <1 2)
- Storage condition of test material: storage at < 40 °C in a tightly closed container under dry conditions (substance very hygroscopic)

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Sampling method: 2 x 20 mL samples were taken from all test solutions and control and at the beginning of the exposure period prior to addition of the algae. After 72 h representative replicates were sampled for chemical analysis. 2 x 20 mL were taken from every test concentration level. One 20 mL sample was frozen as retain sample.
- Sample storage conditions before analysis: samples were acidified for storage. Potassium concentrations were determined within one week after sampling. For more details see section `Details on analytical methods´

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: all stock solutions and the medium were prepared with purified water processed using an ELGA „PURELAB Ultra“. Since the test item was water soluble, the highest test concentration was prepared by adding the required amount of the test item (100.0 mg) to 1 L sterile growth medium under sterile conditions to obtain a nominal concentration of 100 mg/L. The solution was stirred vigorously using a magnetic stirring bar for about 1 h at room temperature (ca. 20 °C). An aliquot of the clear test solution was then be diluted with sterile growth medium to obtain the other 4 test concentrations.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: green algae
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Göttingen, Germany
- Age of inoculum (at test initiation): 3 d
- Stock culture: the stock cultures are maintained fulfilling the criteria of the OECD guideline. The culture medium was recommended by Bringmann und Kühn (Bringmann, G. and Kühn, R. (1980). Comparison of the toxicity thresholds of water pollutants to bacteria, algae, and protozoa in the cell multiplication inhibition test. Water Research 14(3), 231-241.).
- Pre-culture: a pre-culture was established in standard OECD growth medium to obtain exponentially-growing algae for the test

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h

Test conditions

Test temperature:
21.0 °C - 21.5 °C
7.83 - 8.47
Nominal and measured concentrations:
Nominal test item concentrations: control, 6.25, 12.5, 25.0, 50.0 and 100 mg/L test item (corresponding to following nominal potassium concentrations: 1.94, 3.88, 7.75, 15.5 and 31.0 mg K/L)
Measured test item concentrations at test initiation: 1.9, 3.63, 7.27, 15.3, 30.7 mg/L (after background correction)
Details on test conditions:
- Test vessel: 250 mL conical glass flasks covered with air-permeable silicone-sponge caps
- Initial cells density: 10^4/mL
- Control end cells density: 119.63 x 10^4/mL (Std. dev.: 25.8 x 10^4/mL)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8

- Standard medium used: yes, according to guideline

- Source/preparation of dilution water: according to guideline
- Chlorine: NH4Cl 15 mg/L
- Ca/mg ratio: CaCl2 x 2 H2O = 18 mg/L, MgSO4 x 7 H2O = 15 mg/L, MgCl2 x 6 H2O = 12 mg/L
- Intervals of water quality measurement: during the exposure period, the incubation temperature was measured daily with a calibrated thermometer in an additionally prepared control vessel kept under the same conditions. The pH values were measured in the additionally prepared replicate at the beginning of the test and directly in the test vessels at the end of the test.
- Other: the light intensity was measured daily using a cosine (2 π) receptor (LI-250A, LI-COR) in μE m^-2 s^-1 at level of the test media surface

- Photoperiod: continuously illuminated
- Light intensity and quality: 4440 and 8880 lux, day light: OSRAM “day light”
- Other: algae were continuously shaken

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : the cell concentrations were determined in the inoculum culture prior to the addition to the test vessels at test start and after 24, 48 and 72 h in the test cultures
- Determination of cell concentrations: the cell density was measured using an electronic particle counter (CASY® TT, Innovatis, Germany). A calibration curve for relating cell count to fluorescence was prepared. If the cell counts could not be determined using electronic counting or fluorescence measurements, the cells were counted microscopically.

- Range finding study: yes, 72 h, non-GLP, 3 nominal test concentrations of 1.0, 10 and 100 mg test item/L had been tested with stirring the highest test solution for about 1 h with subsequent dilution. Based on visual observations, the test item was soluble at these concentrations.
- Results of range finding test: test item concentrations of 1, 10, 100 mg/L resulted to 2.6, 3.3, 20.1 % growth rate and 14.5, 18.8, 67.6% yield, respectively
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
50 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 37 - 128
72 h
Dose descriptor:
Effect conc.:
25 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes (increase by a factor of 119.6 within the 72 h test period)

Results with reference substance (positive control):
- ErC50: 2.83 mg/L
Reported statistics and error estimates:
The EC50 and EC10 for yield were calculated using non-linear regression. For the determination of EyC50 and EyC10 values for yield a 3-parametric cumulative distribution function (CDF) based on normal distribution of the data was used (Normal-CDF), according to Bruce and Versteeg (Bruce, R.D. and Versteeg, D.J. (1992). A statistical procedure for modeling continuous toxicity data. Environ. Toxicol. Chem. 11, 1485-1494.).

Effective concentrations for growth rate were calculated using linear regression (Probit analysis), since there was a significant lack of fit using nonlinear models (e.g. 3-param. normal cumulative distribution function (CDF)).

Any other information on results incl. tables

The concentrations of the test item in the test media were stable during the test based on the potassium concentrations (94 – 99% of nominal at test start and 96 – 100% of nominal at test end). Therefore, the test evaluation was based on the nominal concentrations of the test item.

Table 1: Measured concentrations of potassium in the test item.

Nominal concentration Measured concentration Measured concentration (minus background)
mg TI/L  mg K/L 0 h mg K/L  72 h aged mg K/L  0 h mg K/L 0 h % nominal  48 h aged mg K/L  48 h aged % nominal
Control  0.459 0.535 0.481 - - - -
6.25 1.94 2.44 2.35 1.9 98 1.87 96
12.5 3.88 4.16 4.2 3.63 94 3.71 96
25 7.75 7.81 8.09 7.27 94 7.61 98
50 15.5 15.8 16 15.3 99 15.5 100
100 31 31.2 30.9 30.7 99 30.4 98
TI = Test Item
K = Potassium

Table 2: Effect concentrations of the test item for the exposure of Pseudokirchneriella subcapitata for 72 h

Parameter*  EC50  EC20  EC10  LOEC  NOEC
Nominal concentrations [mg test item/L]
Growth rate (r)  Value > 100  > 100  n.d.  100 50.0**
95 %-cl lower
95 %-cl upper
Yield (y)  Value  87.3*  28.8*  13.8*  50 25
95 %-cl lower  37 2.06 0.27
95 %-cl upper  128 57.1 34.9
n.d. not determined due to the low inhibition of 15.5% at the highest test concentration
* EC-values were calculated using non-linear cumulative distribution function according to Bruce and Versteeg (1992)).
** Due to the low inhibition of 9.7% at 50 mg/L, the NOEC was set to 50 mg/L, since effects below 10% compared to control are generally not considered to be ecotoxicologically relevant. The statistically significant effect is a result of the low variability of the replicates.

Table 3: Cell number (x 10^4) per mL dependent on test item and time

Treatment nominal [mg test item/L] Control  6.25 12.5 25 50 100
0 h 
1 1 1 1 1 1
24 h 
Replicates: 8 4 4 4 4 4
Mean:  4.852 4.657 5.269 5.165 4.747 4.961
Std.Dev.:  0.47 0.49 0.96 0.47 0.25 0.62
CV:  9.6 10.4 18.2 9 5.4 12.6
48 h 
Replicates: 8 4 4 4 4 4
Mean:  22.19 23.04 22.83 24.39 21.08 18.6
Std.Dev.: 3.1 3.6 3.1 3.9 3.5 2.3
CV: 14.1 15.5 13.8 15.8 16.8 12.6
72 h 
Replicates: 8 4 4 4 4 4
Mean:  119.63 103.88 113.01 104.53 74.37 56.01
Std.Dev.: 25.8 4.2 11.9 10.9 12.1 5.4
CV: 21.6 4.1 10.5 10.4 16.3 9.6
Mean: arithmetic mean; Std. Dev.: standard deviation; n: number of replicates; CV: coefficient of variation

Table 4: Microscopic observation of the cell culture

Date  Observations
Test start, day 0  Inoculum culture: normal appearance of intact cells
Test end, day 3 Control – 25 mg/L Normal appearance of intact cells, almost no cell debris
50 – 100 mg/L Less appearance of intact cells, almost no cell debris

Validity criteria:

The test fulfils the validity criteria of the guideline:

·        The cell number in the control cultures increased by a factor of 119.6 within the 72 h test period (validity criterion: > 16)

·        Evaluation of the sectional growth rates of the controls: the mean of the replicate coefficients of variations (CV %) in the section-by-section growth rate of controls was 6.9% during the test period (validity criterion ≤ 35%)

·        The coefficient of variation of average specific growth rate at test end in replicate control cultures was 4.8% (validity criterion ≤ 7%)

Applicant's summary and conclusion