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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
addaption to the phy-chem properties of the test substance (insoluble in water)
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Carbon black (Printex 30)
IUPAC Name:
Carbon black (Printex 30)
Details on test material:
- Name of test material (as cited in study report): Carbon Black (Printex 30)
- Substance type: furnace black
- Physical state: solid
- Analytical purity: 100%
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition:
- Purity test date:
- Lot/batch No.: K-No. 98040702
- Expiration date of the lot/batch: 31.12.1999
- Radiochemical purity (if radiolabelling):
- Specific activity (if radiolabelling):
- Locations of the label (if radiolabelling):
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions: stable
- Storage condition of test material: at room temperature

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
Carbon black (Printex 30) is insoluble in water, therefore the test substance was tested for toxicity only as aqueous extracts from up to 10,000 mg
test substance /litre (final concentration). For this purpose, 6,250 mg, 630 mg, and 63 mg of the test substance was introduced to 500 ml
ultraoure water by incubation on a shaking machine for 24h at room temperature. During that time an equilibration between the test substance
and the water was considered to be achieved. Thereafter the suspensions were filtered through a filter paper (Schleicher & Schuell 595 N1/2) which
was previously rinsed throughly with ultrapure water in order to eliminate possible soluble impurities from the filter material. Additionally, the
filtered eluates were filtered through sterile membrane filters in order to get sterile conditions for the test. The eluates were used for the test. By
addition of the "intermediate dilution" and the algal inoculation a final nominal concentration of 10,000 mg, 1,008 mg and 100,8 mg test substance was achieved per litre of test solution.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Scenedesmus subspicatus CHODAT
- Strain: No. 86.81
- Source (laboratory, culture collection): Pflanzenphysiologisches Inst. Uni Goettingen [SAG], Sammlung von Algenkulturen
- Age of inoculum (at test initiation):
- Method of cultivation: The algae were purified from accompanying bacteria during the study and further cultivated under strictly aseptic conditions regarding the SOP S 013 of the Inst. of Fresenius
preparation of the algal pre-culture / stock solutions for the "intermediate dilution":
the mineral nutrient medium was prepared from 4 stock solutions as follows:
A: Mineral nutrient salts
1.5 g NH4Cl Merck p.a. Batch 522A992345
1.2 g MgCl2 x 6 H2O Merck p.a. Batch 952A192730
1.8 g CaCl2 x 2H2O Merck p.a. Batch 129TA153982
1.5 g MgSO4 x 7H2O Fluka purum p.a. Analysis -No. 302374191
0.16 g KH2PO4 Merck p.a. Batch A830673448
dissolved in 1 l ultrapure water (Millipore, Milli-Q)
B: Fe-Complex
0.08g FeCl3 x 6H2O Merck p.a. Batch 523B592443
0.10 Na2EDTA x 2H2O Merck p.a. Batch K20894718447
dissolved in 1 l ultrapure water (Millipore, Milli.-Q)
C: Trace elements
185 mg H3BO3 Fluka purum p.a. Analysis-No. 308801/1 392
415 mg MnCl2 x 4H2O Merck p.a. Batch 341A781127
3 mg* ZnCl2 Merck p.a. Batch 141B343815
1.5 mg * CoCl2 x 6H2O Fluka purum p.a. Analysis-No. 290709889
0.01 mg* CuCl2 x 2 H2O Fluka purum p.a. Analysis No. 2679961089
7 mg Na2MoO4 x 2H2O Merck p.a. Batch 005435
dissolved in 1l ultrapure water. To include components marked with *, solutions were prepared by an additional dilution step.
D: sodium hydrogen carbonat
1 g NaHCO3 Merck p.a. Batch K20354929
was introduced in the solid form directly into the "intermediate dilution" which then was sterilized by ultrafiltration. The final concentration of
NaHCO3, was twice as concentrated as indicated in the OECD Guideline 201
Using stock solutions A to D an initial intermediate dilution was prepared as follows:
To 870 ml ultrapure water 100 ml of the stock solution A and 10 ml of each of stock solutions B and C and 1.0g NaHCO3 were added. This solution
was prepared according to the SOP Ö 031 of the Institute Fresenius Group. This solution exhibited a PH of 8.15 after equilibration with air. Prior to
use in the test this "intermediate dilution" was used for the test solutions and the pre-culture to a final concentration of 100 ml/l.


ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not): the same, the algae were held in suspension by stirring on a magnetic stirrer
- Any deformed or abnormal cells observed:
Tree days prior to starting the test, a pre-culture was prepared using the algal stock culture, as followes:
The algal stock culture was diluted to an initial cell concentration of 6.4*10exp5 cells/ml (algal suspension) using the pre-culture medium. This
determination of cell concentration was performed by photometrical measurements (using a calibration curve prepared by using a spectrometer at
578 nm)

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
no

Test conditions

Hardness:
see test solution
Test temperature:
temperature in the incubation chamber was measured daily and documented by the electronic registration system HAMSTER. Values during the main
test were in the range of 21.4 to 24.4 (mean 22.05°C)
pH:
pH = 8.15 after equlibration with air
measuerd by pH meter: WTW portable pH-325 "UBI neu", ser.-no. 7130352
Salinity:
not applicable
Nominal and measured concentrations:
nominal
Details on test conditions:
Suspensions of Printex 30 were prepared in ultrapure water at 6250, 630, and 63 mg per 500 ml batch. After 24 hours incubation at room
temperature on a shaking machine, during which time equilibration was considered to be achieved, the suspensions were filtered through a filter
paper , which had previously been thoroughly rinsed with ultrapure water. These filtered solutions were then passed through sterile membrane
filters to ensure sterile conditThe algal stock culture was cultivated under strictly aseptic conditions within Institut Fresenius. As a functional control, the biomass of the algae in the
control system was shown to increase by more than 16 fold in the 72 hour duration of the test. Three days before the test, a pre-culture was
The sterile test media were prepared from the algal pre-culture, the sterilized filtrates, the mineral medium, and ultrapure water to give nominal
filtrate concentrations from 10,000, 1,008, and 100.8 mg/L Printex 30 suspensions. For the inoculated test solutions with the test substanc, five
parallels were incubated under constant light. A reagent's Blank without algal inoculation three test solutions were prepared as described above
containing a 1:10 diluted intermediate dilution instead of the algal pre-culture. These blanks were incubated under the same conditions under
constant light as the test solution to be tested. The measuerd turbidity in those test solutions was substracted from those with algal inoculation. In
the same way seven control solutions without any test substance but with algal inoculation were prepared as well as one test solution without any test substance and without algal inoculation. By dilution with pre-culture medium the extinction E578nm of the grown preculture was adjusted to a value of 0.20 so that the initial algal concentrations were approximately 8*10 exp4 cells/mL. The incubation of the test and control solutions was
according to OECD test Guideline 201, for a period of 72 hours at 21.4 to 24.4 deg C (mean 22.05), at an illumination rate of greater than
120 µE/m2s. The solutions were stirred for 15 minutes in every hour with a magnetic stirrer, to ensure adequate CO2 input.
The increase in biomass was determined using a spectrophotometer after 24.5h, 49h, 72h.. As a mesure for the biomass the extinction of the test
solutions measured at 578 nm, was used. The test was performed under aseptic conditions (with the exeption that the stock solutions of the test
substance were not autoclaved or sterilized by membrane filtration. For this purpoes the test vessels were sterilized by heat (3h at 180°C). The ultrapure water used for the test solutions were sterilized by autoclaving. During incubation the test solutions were covered with a Plexiglas lid, to maintain
sterile conditions.
As a first step a screening test was performed using 10,000 mg/l, 1,008 mg/l and 100,8 mg/l (final concentrations) in order to get information on
the concentration range to be used within the main test. As the test substance exhibited no toxic effects during the screening test no further main
test was performed.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Under the conditions of the test, the test substance exhibit no chronic toxic effect towards the test system/ Scenedesmus subspicatus. The inhibition of the biomass and growth rate (EC10 and EC50) was defined as being the aqueous extract from > 10, 000 mg/l.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, the test substance exhibit no chronic toxic effect towards the test system/ Scenedesmus subspicatus. The inhibitionof the biomass and growth rate (EC10 and EC50) was defined as being the aqueous extract from > 10, 000 mg/l.
Executive summary:

Carbon black was not toxic within its range of aqueous solubility.

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