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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
23rd March, 2006
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Test material form:
solid: crystalline

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Vehicle:
yes
Remarks:
water

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Raphidocelis subcapitata
(formerly known as Pseudokirchneriella subcapitata)
Strain number: 61.81 SAG
(identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Origin: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
Breeding Conditions: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardized conditions according to the test guidelines.
Pre-culturing: The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of 2-4 days. (The pre-culture was incubated for four days at this test.) The algal cultures used in this study did not contain deformed or abnormal cells.

Study design

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

Test conditions

Hardness:
na ( rconstituted algae growth medium)
Test temperature:
Temperature: Culture temperature was checked at the beginning of the study and each day thereafter in a flask filled with water, in the climatic chamber. In addition temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was between 22.4 and 22.8°C measured in the flask and in the range of
21.6 – 23.8°C measured within the climate chamber.
pH:
pH: The pH was measured in each test group at the start (before test solutions had been distributed into the test vessels) and in each test vessel at the end of the test. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.58 – 9.39 during the experiment.
Dissolved oxygen:
na
Salinity:
na
Conductivity:
na
Nominal and measured concentrations:

Nominal
concentration
[mg/L] Measured concentration [mg/L] Geometric mean
[mg/L]
0-h 24-h 48-h 96-h
0.7 0.76 0.25 0.25 0.25 0.33
1.9 1.88 0.25 0.25 0.25 0.41
5.1 4.92 0.25 0.25 0.25 0.53
13.7 13.4 0.25 0.25 0.25 0.68
37.0 35.7 11.6 0.25 0.65 2.86
100.0 96.3 81.7 0.25 2.38 8.27

Details on test conditions:
Light Intensity: The light intensity was checked and recorded at the start of the test. The algal culture flasks were continuously illuminated. The average light intensity measured at the position occupied by algal culture flasks during the test was 7581 lux, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.
Reference substance (positive control):
yes

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
>= 8.27 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.:
ca. 0.68 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.:
ca. 1.94 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.68 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item Ammonium Adipate had inhibitory effects on the growth of Raphidocelis subcapitata.
All validity criteria were met. The results are based on measured geometric mean test item concentrations.

Biological endpoints of the study are summarised below:

Summary of the Biological Endpoints (same as Table 1)
Endpoints
(0-72 h) Growth rate (µ)
[mg/L] Yield (y)
[mg/L]
Calculation based on measured concentrations
EC10 1.35 0.50
95 % conf. limits 0.99 – 1.73 0.33 – 0.68
EC20 2.65 0.80
95 % conf. limits 2.07 – 3.40 0.57 – 1.05
EC50 >8.27 (9.65 calculated) 1.94
95 % conf. limits 6.97 – 15.10 1.47 – 2.70
NOEC 0.68 0.68
LOEC 2.86 2.86

Executive summary:

Test item:

Ammonium Adipate(Lot No.:N118031457)

Test species:

Raphidocelis subcapitata(formerly known asPseudokirchneriella subcapitata);Strain number:61.81 SAG

Exponentially-growing cultures; preculture was incubated for four days before the test.

Dilution water:

OECD medium, prepared inthe laboratory of TOXI-COOP ZRT.

Concentrations:

Nominal concentrations of 0.7, 1.9, 5.1, 13.7, 37.0 and 100.0 mg/L were investigated in the main study. Concurrent control ran.

The measured concentrations deviated more than 20 % from the nominal during the experiment (as the test item was not stable in OECD medium) therefore the geometric mean of the measured concentrations were calculated to determine exposure concentrations.

The corresponding calculated geometric mean concentrations were the followings: 0.33, 0.41, 0.53, 0.68, 2.86 and 8.27 mg/L.

Biological results and endpoints are based on the measured geometric mean concentrations.

Test design:

Exponentially-growing cultures ofRaphidocelis subcapitatawere exposed to the test item under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 104 cells/mL at the start of the test in all of the test cultures. Glass flasks with total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals.

Analytics:

For determination of the test item concentrations,samples were taken from each concentration level and control atthe start and at the end of the test. Concentrations were determined usingHPLC method with UV detection.


Endpoints:

EC10, EC20, EC50, NOEC and LOEC for both response variables (i.e. average specific growth rate and yield).

Statistics:

For the determination of the LOEC and NOEC, ANOVA and Dunnett’s test (Dunnett-t and Dunnett T3,a= 0.05) for determination of the ECxvalues Probit analysis was used.


Validity:

All validity criteria were met and therefore the study can be considered as valid (see section 7.1)

 


 

Results:

All biological results are based on the measured geometric mean concentrations.

The biological endpoints are summarised below in Table 1.

 

Table 1:Summary of the biological endpoints

Endpoints
(0-72 h)

Growth rate (µ)
[mg/L]

Yield (y)
[mg/L]

Calculation based on measured concentrations

EC10

1.35

0.50

95 % conf. limits

0.99 – 1.73

0.33 – 0.68

EC20

2.65

0.80

95 % conf. limits

2.07 – 3.40

0.57 – 1.05

EC50

>8.27(9.65 calculated)

1.94

95 % conf. limits

6.97 – 15.10

1.47 – 2.70

NOEC

0.68

0.68

LOEC

2.86

2.86