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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
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
- Sampling method:
- Sample storage conditions before analysis:
Vehicle:
no
Details on test solutions:
The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system was prevented as much as possible (e.g. film of the test substance on the water surface).

The batch of MEA Polyborate 1:1 tested was a clear colourless liquid composed of 40% of salt in water and completely soluble in test medium at the concentrations tested.

Preparation started with a concentration of 32 mg active ingredient (a.i.)/l which corresponds to 80 mg MEA Polyborate 1:1 per litre. To this end 63 µl of the test substance was added to 1000 ml test medium and was magnetically stirred for a period of 15 minutes. The pH of the highest concentration was adjusted from 9.3 to 8.0 with 1M HCl (Merck, Darmstad, Germany). The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.

After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 104 cells/ml.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Fresh water alga, Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In house laboratory culture
- Age of inoculum (at test initiation):
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
22.7°C – 23.3°C
pH:
8.1 - 8.3
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal: 0.8, 2.5, 8.0, 25, 80 mg MEA Polyborate 1:1 per litre
Measured: 0.3, 1.0, 3.2, 10, 32 mg MEA Polyborate 1:1 per litre
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml, all glass, containing 50 ml of test solution
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 1 x 104 cells/mL

GROWTH MEDIUM
- Standard medium used: M2 according to OECD 201 Guideline
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
NH4Cl 15 mg/l
MgCl2.6H2O 12 mg/l
CaCl2.2H2O 18 mg/l
MgSO4.7H2O 15 mg/l
KH2PO4 1.6 mg/l
FeCl3.6H2O 64 µg/l
Na2EDTA.2H2O100 µg/l
H3BO3 185 µg/l
MnCl2.4H2O 415 µg/l
ZnCl2 3 µg/l
CoCl2.6H2O 1.5 µg/l
CuCl2.2H2O 0.01 µg/l
Na2MoO4.2H2O7 µg/l
NaHCO3 50 mg/l
Hardness (Ca+Mg) 0.24mmol/l (24 mg CaCO3/l)
pH 8.1 ± 0.2


OTHER TEST CONDITIONS
- Light intensity and quality: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 81 to 88 E.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]:At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with a cuvette (pathlength =10 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.

TEST CONCENTRATIONS
- Test concentrations: 0.8, 2.5, 8.0, 25, 80 mg MEA Polyborate 1:1 per litre
Reference substance (positive control):
yes
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
26 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 21 - 31 mg a.i./L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 5.2 - 30 mg a.i./L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of a GLP OECD 201 study with Pseudokirchneriella subcapitata, MEA Polyborate 1:1 reduced growth rate and inhibited the yield of this fresh water algae species significantly at 10 mg a.i./l and higher.

The EC50 for growth rate reduction (ERC50: 0-72h) was 26 mg a.i./l with a 95 % confidence interval ranging from 21 to 31 mg a.i./l.

The EC50 for yield inhibition (EYC50: 0-72h) was 13 mg a.i./l with a 95 % confidence interval ranging from 5.2 to 30 mg a.i./l.

The NOEC for growth rate reduction and yield inhibition was 3.2 mg a.i./l.
Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition with

MEA Polyborate 1:1.

 

The study procedures described in this report were based on the OECD guideline No. 201, 2006. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 761/2009and the ISO International Standard 8692, 2004.

 

The batch of MEA Polyborate 1:1 tested was a clear colourless liquid composed of 40% of salt in water and completely soluble in test medium at the concentrations tested.

 

A final test was performed based on the results of a range-finding test. Preparation started with a concentration of 32 mg active ingredient (a.i.)/l which corresponds to 80 mg MEA Polyborate 1:1 per litre. To this end 63 µl of the test substance was added to 1000 ml test medium and was magnetically stirred for a period of 15 minutes. The pH of the highest concentration was adjusted with 1M HCl. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium.

 

Exponentially growing algae were exposed to a control (six replicates) and concentrations of 0.32, 1.0, 3.2, 10 and 32 mg a.i. (three replicates each). The initial cell density was 104cells/ml and the total test period was 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 hours of exposure and at the end of the test.

 

Analyses were performed on samples taken from the nominal concentrations of 0.8, 2.5, 8.0, 25 and 80 mg/l (i.e. 0.32, 1.0, 3.2, 10 and 32 mg a.i./l). The concentrations of both borate and MEA were determined.

 

Borate

The corresponding nominal concentrations of boron in analysed samples were 52, 162, 518, 1618 and 5176 µg borate per litre, respectively.

Analysis of the samples taken at the start of the final test showed that the actual concentrations were in agreement with nominal, 78 - 94% of nominal. The measured concentrations remained stable during the entire test period (101 - 119% of initial).

 

MEA

The corresponding nominal concentrations of MEA in analysed samples were 0.16, 0.5, 1.6, 5.0 and 16 mg MEA per litre, respectively.

Analysis of the samples taken at the start of the final test showed that the actual concentrations were in agreement with nominal, i.e 78 - 101% of nominal and remained stable during the entire test period (90 - 117% of initial)

 

Based on these results, the effect concentrations were expressed in terms of the nominal concentrations of the active ingredient.

 

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

MEA Polyborate 1:1 reduced growth rate and inhibited the yield of this fresh water algae species significantly at 10 mg a.i./l and higher.

 

The EC50for growth rate reduction (ERC50: 0-72h) was 26 mg a.i./l with a 95 % confidence interval ranging from 21 to 31 mg a.i./l.

The EC50for yield inhibition (EYC50: 0-72h) was 13 mg a.i./l with a 95 % confidence interval ranging from 5.2 to 30 mg a.i./l.

The NOEC for growth rate reduction and yield inhibition was 3.2 mg a.i./l.

Description of key information

Under the conditions of a GLP OECD 201 study with Pseudokirchneriella subcapitata, MEA Polyborate 1:1  reduced growth rate and inhibited the yield of this fresh water algae species significantly at 10 mg a.i./l  and higher.

The EC50 for growth rate reduction (ERC50: 0-72h) was 26 mg a.i./l  with a 95 % confidence interval ranging from 21 to 31 mg a.i./l.

Key value for chemical safety assessment

EC50 for freshwater algae:
26 mg/L

Additional information