Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb 2012 to May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD 201 guideline study under GLP
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
10 ml samples were taken from the analytical parallels at T=0 and subsequently from both the test replicates and analytical parallels at 24, 48 and 72 hours.
Vehicle:
no
Details on test solutions:
The test chemical was known to dissolve readily in the test media, thus no special solvents or dissolution techniques were used.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Source and maintenance of algae
The test was carried out with the freshwater unicellular algae P. subcapitata (CCAP 278/4) obtained from the Culture Collection of Algae and Protozoa, Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland, UK. After purchasing this strain was cultured and maintained. Cultures on sloped agar tubes were stored at 4°C until required. Exponentially growing cultures are maintained at 23 ± 2°C in a temperature-controlled illuminated orbital incubator and are re-cultured under sterile conditions weekly to keep the algae in this phase. For the evaluation of the quality of the algae and the experimental conditions, the reference substance potassium dichromate was tested at least twice a year to demonstrate satisfactory test conditions and algae sensitivity

Preparation of the inoculum
The initial stock culture was inoculated with P. subcapitata from a sloped agar tube and checked for purity by microscopic means. This algal stock culture (40 mL) of P. subcapitata was regularly transferred to fresh medium to act as inoculum for testing. The absorbance of an exponentially growing stock culture was measured. The cell density was determined using the calibration curve described below. From this algal culture a dilution was prepared to obtain an initial cell density of approximately 1104 cells/mL in each of the test vessels.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
NaHCO3: 150 mg/L
Test temperature:
22.85 to 23.65 °C
pH:
8.1 to 8.3
Dissolved oxygen:
not relevant
Salinity:
not relevant
Nominal and measured concentrations:
Nominal (mg/L) 0.35, 1.12, 3.58, 11.5, 36
Measured
Details on test conditions:
Test flasks
The test was performed in sterile 100 mL Erlenmeyer flasks with cotton wool stops to allow gas exchange. The test vessels were filled to a total of 40 ml with mineral media / test substance.

Culturing cabinet and test conditions
The test was carried out in a temperature-controlled illuminated orbital incubator in which the temperature was maintained at 23 ± 2°C. Uniform Illumination was provided in the spectral range of 400 to 700 nm by using fluorescent lamps at a distance of about 0.36  0.02 m from the algal cultures. The light intensity was in the range of 60 to 120 µE•m-2•s. The test vessels were agitated continuously at a speed sufficient to prevent sedimentation of the algae (100 rpm approx).

Preparation of stock solutions
The test chemical was known to dissolve readily in the test media. A stock of approximately 100 mg/L was made by accurate weighing of the test chemical, addition and subsequent moderate to fast stirring until a homogeneous solution was achieved. The pH was then adjusted slightly from 8.7 to 8.1 with 0.1 M HCL. The test chemical was then pipetted (whilst the stirring the stock solution) in the correct volumes to the test vessels as previously described to generate the test concentrations.

Test concentrations
The following test concentrations in triplicate including 6 controls were tested:
0.35, 1.12, 3.58, 11.5 and 36.0 mg/L. An additional replicate of the same concentrations without test organisms was also prepared for analytical purposes.


Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
6.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Results with reference substance (positive control):
The ErC50 value of the reference compound, potassium dichro¬mate, was in the range of 0.25-2.0 mg/L (Documented as part of GLP laboratory maintenance).
Reported statistics and error estimates:
EC10 and EC50 values were calculated by a maximum likelihood probit plot. NOEC and LOEC were also determined using a Dunnetts test. Normal distribution and homogeneity could not be confirmed be confirmed by the Shapiro-Wilks and the Bartlett’s tests however. The statistical test used has shown to be durable to heterogeneous data (Ref 6) however the ErC¬10 may be used as an acceptable alternative to the NOEC if required.

Quality criteria for the analytical method

Validation

Parameter

Result

Requirement

Linearity       

R squared

R2=0.997
(lowest value)

R2≥ 0.98

Reproducibility
(0.1 mg/L)

Coefficient of variation

9.8%

< 20%

Reproducibility
(10 mg/L)

Coefficient of variation

5.2%

< 20%

Limit of quantification

LOQ

0.04 mg/L
(highest value)

< 1mg/L

Limit of detection

LOD

0.01 mg/L
(highest value)

< 1 mg/L

Recovery from medium

-

>72.2% (worst case)

>60 <120%

System stability

Difference control stand. from expected value

-7.9 to 9.8%

< 10%

Validity criteria fulfilled:
yes
Remarks:
cell density of the controls increased at least a factor 16 within 72 h, mean coeff. of variation for section-by-section specific growth rates in the control did not exceed 35 %, EC50 value of the reference compound, KCr207, was in the range 0.25-2.0 mg/L
Conclusions:
The ErC10 (72h) was calculated as 2.9 mg/l (Growth rate). The ErC50 (72h) was calculated as 5.6 mg/l. (Growth rate Geometric Measured Means).
Executive summary:

Summary

In order to predict the effects of1,1,3,3-Tetramethylbutyl hydro peroxidein an aquatic environment, an Algal Growth Inhibition test was conducted in accordance with OECD test guidelines and with the OECD Principles of Good Laboratory Practice.  

The toxicity ofthe test chemicalto an exponentially growing culture ofP. subcapitatawas determined in a sealed system over an exposure period of 72 hours. Nominal loading concentrations of0.35, 1.12, 3.58, 11.5 and 36.0 mg/Lwere tested. For the highest 3 concentrationsa statistically significant effect in growth rate in comparison to the control was found.

The ErC10(72h) was calculated as 2.9 mg/L (Growth rate). The ErC50(72h) was calculated as 5.6 mg/L. (Growth rate Geometric Measured Means).

 

TheNOECwas determined as 2.0 mg/L (Growth rate) and the LOEC was determined as 6.9 mg/L (Growth rate Geometric Measured Means).

 

Values are reported as geometric means of measured concentrations in parallel replicates during the test without test organisms. Analytical measurements in the actual replicates were conducted however due to the reactive (oxidising) nature of the test chemical. The test chemical was degraded in the test replicates containing algal growth and was therefore not measurable.

 

The following quality criteria have been met in the present study:

 

·        The cell density of the controls increased at least a factor 16 within 72 hours. .

·        The coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures did not exceed 7% (See Table 4 a).

·        The ErC50value of the reference compound, potassium dichro­mate, was in the  range of 0.25-2.0 mg/L (Documented as part of GLP laboratory maintenance). 

·          The mean coefficient of variation for section-by-section specific growth rates in the control did not exceed 35 %

·          The validity criteria for the analytical method were met.

 

The following criterion was not met:

 

·          The test concentration did not stay within 80 and 120 % of the nominal concentrations. Geometric means of the measured concentrations were therefore used for endpoint calculations.

 

 

Chemical analyses showed that the concentration of the test chemical steadily decreased during the test duration and was not maintained at > 80% of the nominal concentration. For this reason geometric means of the measured concentrations were calculated and used for expressing of the endpoints. 

 

Description of key information

One OECD 201 GLP study is available with a  ErC10 (72h) of  2.9 mg/l  and ErC50 of 5.6 mg/l

Key value for chemical safety assessment

EC50 for freshwater algae:
5.6 mg/L
EC10 or NOEC for freshwater algae:
2.9 mg/L

Additional information

One OECD 201 GLP study with analytical monitoring is available which meets all the quality criteria. As the test substance did not remain stable during the test, the effect values were calculated based on geometric mean values.