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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not specified
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
Toxicity to bacteria (Pseudomonas putida) and blue algae (Microcystis aeruginosa) was assessed following a growth inhibition test method.
GLP compliance:
not specified
Test material information:
Composition 1
Analytical monitoring:
not specified
Details on test solutions:
The substance was dissolved in distilled water, pH 7
Test organisms (species):
other: bacteria (Pseudomonas putida) and blue algae (Microcystis aeruginosa)
Test type:
not specified
Water media type:
not specified
Details on test conditions:
Re-culture media and culture conditions for stock cultures, pre-cultures and test cultures of the test organisms were all standardised.
Cell seed, cell proliferation or inhibition of cell proliferation were quantified by turbidimetric monochromatic measuring radiation, shielding the scattered light and compensating for the absorption of the primary light by dissolved substances.
Key result
Dose descriptor:
other: TGK
Effect conc.:
13 mg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
not specified
Remarks on result:
other: Microcystis aeruginosa
Key result
Dose descriptor:
other: TGK
Effect conc.:
330 mg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
not specified
Remarks on result:
other: Pseudomonas putida
Details on results:
The evaluation of the results of the measurements was identical for both model organisms and corresponded to the following scheme:
For the graphic evaluation of the toxicological findings after the end of the test period, the measured values of the extinction of the test culture were summarised by three homologous dilution series, calculated, as follows:

(A) the mean value of the extinction of all test cultures with non-toxic pollutant concentrations,
(B) the mean value of the extinction of the three test cultures of the highest non-toxic pollutant concentrations,
(C) the mean extinction of the three test cultures with the lowest toxic pollutant concentrations

The range of non-toxic pollutant concentrations is defined by a standard deviation of the extinction value <3%. The mean extinction of all test cultures with non-toxic concentrations (A) is reduced by the extinction difference 3% in the absorbance / pollutant concentration coordinate system as reference horizontal. The mean extinction of the test cultures with the highest non-toxic nitrogen concentration (b) is then above the horizontal reference, the mean extinction of the test cultures with the lowest toxic pollutant concentration (C) below the reference horizontal. The intersection point of the B-to-C connection line with the reference horizon indicates the pollutant concentration of the onset inhibition of cell proliferation for pseudomonos putida or Microcystis aeruginosa, respectively. If a negative deviation of the mean extinction value around the extinction differential is 3% of the mean extinction of all test cultures with non-toxic pollutant concentrations is used as an indicator of the incipient damaging effect.
Validity criteria fulfilled:
not specified
Conclusions:
Under the conditions of the study the TGKmi was determined to be 13 mg/L, and the TGKps was determined to be 330 mg/L. The TGKmi : TGKps was 1:25.
Executive summary:

The incipient inhibition of water-endangering substances was tested on the cell proliferation of both the bacterium Pseudomonas putida and the blue-green algae Microcystis earuginosa, and the respective concentration of harmful substances (toxic concentration = TGK) causing cell inhibition was determined for each of the two test organisms. From this, the ratio of the toxic limit concentration of each tested substance for the blue-algae Microcystis (TGKmi) to the toxic limit concentration of the corresponding substance for the bacterium Pseudonomas (TGKps) was calculated, with TGKmi = 1 being set.

Under the conditions of the study the TGKmi was determined to be 13 mg/L, and the TGKps was determined to be 330 mg/L. The TGKmi : TGKps was 1:25.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
190 substances were assessed by means of the cell multiplication inhibition test in standard media using the bacteria (Pseudomonas putida) and green algae (Scenedesmus quadricauda).
GLP compliance:
not specified
Test material information:
Composition 1
Details on test solutions:
The substance was dissolved in distilled water, pH 7.0
Test organisms (species):
other: bacteria (Pseudomonas putida) and green algae (Scenedesmus quadricauda)
Details on test organisms:
TEST ORGANISM
- Pseudomonas putida
Strain cultures of the test strain Pseudomonas putida were kept in culture tubes on the nutrient medium for stem and pre-culture. For the further cultivation of the test strain, new stem carvings were created within 1 week. The inoculated stock cultures were incubated for 24 hours at 25°C and kept in stock.
As required, pre-cultures were placed in culture tubes and incubated at 25°C for 24 hours. Thereafter, the cell material was washed off with sterile NaCl solution. For the bacterial suspension, the extinction of the monochromatic measuring radiation Hg 436 nm, for a thickness of 10 mm, was determined by photoelectric measurement.

- Scenedesmus quadricauda
Stem cultures of Scenedesum quadricauda were stored in 100 mL Erlenmeyer flasks, which were sealed with metal caps, in 20 mL nutrient solution, on a white base under exclusion of daylight with continuous illumination in the midfield of two parallel fluorescent lamps (mutual luminaire spacing 60 cm) at 27°C and 50% rel. humidity. For further cultivation of the test strain, new stem cultures were continuously created within 10 days. For this purpose, a presumed number of erlenmeyer flasks, which are closed with metal caps, were sterilised in a steam pot with 20 mL of nutrient solution every 30 minutes on two successive days. After cooling, the contents of each collagen are inoculated with 2 mL of cell suspension from a 10 day old strain culture.
Pre-cultures were prepared in the same manner and kept for 10 days under the same conditions as the stock cultures
Details on test conditions:
TEST MEDIA
Pure cultures of the unicellular model organisms were used as test organisms. The culture media and culture conditions for stock cultures, pre-cultures and test cultures of the model organisms were standardised. Cell seed, cell proliferation or inhibition of cell proliferation were quantified turbidimetrically from the extinction of the primary light of the standardised monochromatic measuring radiation, shielding the scattered light and compensating for the absorption of the primary light by dissolved substances.

- Determination of the biological effect of water-polluting substances against Pseudonomas putida
Each test flask contained 80 mL of culture fluid. The filling of each test flask of the three rows of inoculations to be inoculated to the desired value of 100 mL was then carried out by adding 5 mL each of the two stock solutions and 10 mL of the prepared bacterial suspension of the pre-culture having a known extinction value. The filling of each test flask of the unimpregnated dilution series to the desired value of 100 mL was carried out by adding 5 mL of each of the two stock solutions and 10 mL of NaCl solution.
Inoculated and control vessels were maintained at 25°C for 16 hours. At the end of the test period, the extinction of the monochromatic measuring radiation Hg 436 nm for a layer thickness of 10 mm was measured in the inoculated dilution series.

- Determination of the biological effect of water-polluting substances against Scenedesmus quadricauda
Each test flask contained 40 mL of culture fluid. The filling of each flask of the dilution series to be inoculated to the desired value of 50 mL was then carried out by adding 5 mL of the stock solution and 5 mL of the algae suspension of the pre-culture having a known adjusted extinction value. The filling of each flask of the control dilution series to the target value of 50 mL took place by adding 5 mL of stock solution and 5 mL of distilled water.
The culture tubes were capped with metal caps and shaken. The filled culture tubes of the dilution series were kept for 8 days under the same conditions as the stock cultures and shaken once a day. After the test period had elapsed, the cultures were shaken to obtain a uniform cell suspension immediately before the measurement. Then the extinction of the monochromatic measuring radiation Hg 578 nm of the cell suspension of each test culture for 10 mm layer thickness is measured.
Reference substance (positive control):
not specified
Key result
Dose descriptor:
other: TGK
Effect conc.:
330 mg/L
Nominal / measured:
not specified
Remarks on result:
other: Pseudomonas putida
Key result
Dose descriptor:
other: TGK
Effect conc.:
11 mg/L
Nominal / measured:
not specified
Remarks on result:
other: Scenedesmus quadricauda
Details on results:
The toxic limit concentration (TGK) of the substance for Pseudomonas putida was reported to be: 330 mg/L
The toxic limit concentration (TGK) of the substance for Scenedesmus quadricauda was reported to be: 11 mg/L
Validity criteria fulfilled:
not specified
Conclusions:
Under the conditions of the study, the toxic limit concentration (TGK) of the substance for Pseudomonas putida was reported to be 330 mg/L and the toxic limit concentration (TGK) of the substance for Scenedesmus quadricauda was reported to be 11 mg/L.
Executive summary:

Using analogous methods of the cell multiplication inhibition test, the toxicity threshold (TGK) of 190 potential pollutants was determined for bacteria (Pseudomonas putida) and green algae (Scenedesmus quadricauda).

Under the conditions of the study, the  toxic limit concentration (TGK) of the substance for Pseudomonas putida was reported to be 330 mg/L and the toxic limit concentration (TGK) of the substance for Scenedesmus quadricauda was reported to be 11 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: DIN 38 412, Part 9 (draft standard)
GLP compliance:
not specified
Test material information:
Composition 1
Details on test solutions:
PREPARATION OF TEST SOLUTION
In order to produce the test material, water-soluble substances were quantitatively dissolved to produce an optically clear stock solution in 800 mL double-distilled water using magnetic stirrers (maximum 24 h).
In order to produce the test material of very poorly soluble substances, an attempt was also made to dissolve them quantitatively in 800 mL double-distilled water using magnetic stirrers. Optically unclear solutions were filtered through fibre-glass filters and the filtrate was quantified chemically. The results obtained were expressed as the arithmetically calculated concentrations of the dilution steps, based on the chemically, analytically determined concentration in the filtrate of the stock solution.
In some cases it was necessary to dissolve the substance in a relatively high concentration in pure ethanol (unmethylated) so that only volumes in the microlitre range had to be transferred to the double-distilled water for the production of the stock solution.
Before beginning the test, the stock solutions of the substances to be tested (sample) were adjusted to pH 8.0 ± 0.3 whereby the concentration of the acid or base was selected so as to ensure that the volume to be added remained small.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: 8681 SAG

STOCK CULTURES
The laboratory strain had been maintained over a number of decades in submerged culture.
For this purpose, the required number of 100 mL Erlenmeyer flasks with metal caps containing 20 mL nutrient solution was sterilised in a steam steriliser for 30 min on 2 consecutive days. After cooling, the contents of each flask were inoculated with 2 mL cell suspension taken from a 10-day old stock culture.
The inoculated flasks were placed on a white surface, protected from daylight and exposed to constant lighting from two parallel fluorescent Osram 40 W/30 tubes (distance from each tube 60 cm: irradiance E0, Sy = 24.9 W/m ²) at 24 ± 1°C and relative humidity of 50%. To maintain the test strain, fresh stock cultures were prepared at 10-day intervals.

PRELIMINARY CULTURE
The cultivation of the preliminary cultures was undertaken 3 days prior to the preparation of the test solution. For this purpose, 50 mL nutrient solution (for test cultures) were filled into 300 mL Erlenmeyer flasks with metal caps and inoculated with Scenedesmus from 7-day-old stock cultures. The cell concentration in the preliminary culture flasks must amount to 10^4 per mL. This was to ensure that the culture was still in a process of logarithmic growth after 72 h. Light and temperature conditions corresponded to those for the test preparation. The cell material of the preliminary cultures was used after 72 h to inoculate the dilution preparation after the cell concentration had been fixed at 10^5 per mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
pH:
8 ± 0.3
Nominal and measured concentrations:
3.9 - 500 mg/L
Details on test conditions:
TEST PREPARATION
From the test preparation and from sterile double-distilled water as the diluent, dilution series with various volume ratios were set up. Dilution steps with a dilution series of 1 : 2 (f = 2) each had 1 part of pollutant solution in 2^0 to 2^8 volumes of mixture.
300 mL Erlenmeyer flasks with metal caps were used as the test vessels. The procedure was as follows: using a 1000 mL flat-bottom flask with 800mL pollutant (test preparation) of the highest concentration to be tested, the further dilution steps were each set up in a constant dilution ratio according to the pattern: 400mL preliminary dilution of the test medium + 400mL dilution water. Four hundred mL fluid were thus added to each of the flasks in the dilution series. By adding 50 mL of the 10-fold concentration of the nutrient solution for the test preparation (DIN Test Procedure L9) and 50 mL of the algal suspension of the preliminary culture (10^5 cells per mL), the desired theoretical value of 500 mL was reached. The inoculum in each flask now corresponded to a cell concentration of 10^4 per mL.
Whilst shaking the contents, 50 mL were removed from each concentration step in eight 300 mL Erlenmeyer flasks and the flasks closed with metal caps. Thus, on each measurement day two flasks from each concentration level of the dilution series were available for measuring.
Control preparations (with no test material) were prepared from dilution water, nutrient solution and algal suspension from the preliminary culture in eight 300mL Erlenmeyer flasks and treated in the same way as the test preparation.

MEASURING
The extinction value of the monochromatic radiation (578 nm) of the cell suspension was determined for each concentration level of the test and control preparations after 24, 48, 72 and 96 h. The method selected to determine the biomass was measurement of optical density (measurement of turbidity). The equipment used was the Eppendorf digital photometer 6115 S with a facility to measure turbidity, filter 578 nm, (1 or 2 cm cell flask).
After determining the optical density, the pH-value was measured in each test and control vessel.

EVALUATION
The data were evaluated in line with the Standard. The following procedure was applied to each substance:
- the establishment of growth curves for each tested concentration of the test material and for the control;
- the area under the growth curves (integral of the biomass with time) was calculated according to the given equation and from this was calculated the "percentage inhibition of cell multiplication (Hb) during the test for each tested concentration level of the test preparation on the basis of a comparison of the biomass formed under the influence of the substance (Br) with the biomass in the control preparation (Bk).
- the average growth rate for cultures showing exponential growth was calculated, the basis being the time between the beginning of the test and 72 h later. On this basis the growth-related inhibition (Hµ) was calculated from the difference between the growth rates (µk - µr) and the growth rate µK in the control preparation;
- the tested concentration was assigned to the respective inhibition values in the probability paper. The regression line was determined and from this could be read the desired values EbC10, EbC50 and/or EµC10 and EµC50.
Reference substance (positive control):
not specified
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
240 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Conclusions:
Under the conditions of the study the 72 hour EC50 (growth rate) of the registered substance was > 500 mg/L and the 72 hour EC10 (growth rate) was > 500 mg/L. The 72 hour EC50 (biomass) was > 500 mg/L and the 72 hour EC10 (biomass) was 240 mg/L.
Executive summary:

In the Scenedesmus subspicatus cell multiplication inhibition test, 68 potentially hazardous substances were examined to determine the effect concentrations (EC). The tests were conducted in accordance with the test procedure DIN 38 412, Part 9 (draft standard). The green alga Scenedesmus subspicatus was cultivated as the test organism.

Under the conditions of the study the 72 hour EC50 (growth rate) of the registered substancewas > 500 mg/L and the 72 hour EC10 (growth rate) was > 500 mg/L. The 72 hour EC50 (biomass) was > 500 mg/L and the 72 hour EC10 (biomass) was 240 mg/L.

Description of key information

Kuhn (1990)

Under the conditions of the study the 72 hour EC50 (growth rate) of the registered substance was > 500 mg/L and the 72 hour EC10 (growth rate) was> 500 mg/L. The 72 hour EC50 (biomass) was > 500 mg/L and the 72 hour EC10 (biomass was 240 mg/L.

Bringmann (1976)

Under the conditions of the study the TGKmi was determined to be 13 mg/L, and the TGKps was determined to be 330 mg/L. The TGKmi : TGKps was 1:25.

Bringmann (1977b)

Under the conditions of the study, the  toxic limit concentration (TGK) of the substance for Pseudomonas putida was reported to be 330 mg/L and the toxic limit concentration (TGK) of the substance for Scenedesmus quadricauda was reported to be 11 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
500 mg/L
EC10, LC10 or NOEC for freshwater algae:
500 mg/L

Additional information

Kuhn (1990)

In the Scenedesmus subspicatus cell multiplication inhibition test, 68 potentially hazardous substances were examined to determine the effect concentrations (EC). The tests were conducted in accordance with the test procedure DIN 38 412, Part 9 (draft standard). The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997) and was selected as the key study for this endpoint.

Under the conditions of the study the 72 hour EC50 (growth rate) was > 500 mg/L, the 72 hour EC10 (growth rate) was > 500 mg/L. The 72 hour EC50 (biomass) was > 500 mg/L, the 72 hour EC10 (biomass was 240 mg/L.

Two supporting studies are available, both were awarded a reliability score of 4 in accordance with the criteria set forth by Klimisch et al. (1997):

Bringmann (1976)

The incipient inhibition of water-endangering substances was tested on the cell proliferation of both the bacterium Pseudomonas putida and the blue-green algae Microcystis earuginosa, and the respective concentration of harmful substances (toxic concentration = TGK) causing cell inhibition was determined for each of the two test organisms. From this, the ratio of the toxic limit concentration of each tested substance for the blue-algae Microcystis (TGKmi) to the toxic limit concentration of the corresponding substance for the bacterium pPeudonomas (TGKps) was calculated, with TGKmi = 1 being set.

Under the conditions of the study the TGKmi was determined to be 13 mg/L, and the TGKps was determined to be 330 mg/L. The TGKmi : TGKps was 1:25.

Bringmann (1977b)

Using analogous methods of the cell multiplication inhibition test, the toxicity threshold (TGK) of 190 potential pollutants was determined for bacteria (Pseudomonas putida) and green algae (Scenedesmus quadricauda).

Under the conditions of the study, the  toxic limit concentration (TGK) of the substance for Pseudomonas putida was reported to be 330 mg/L and the toxic limit concentration (TGK) of the substance for Scenedesmus quadricauda was reported to be 11 mg/L.