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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:
key study
Study period:
11 Feb 2013 to 22 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was conducted in accordance with international guidelines (OECD 201), and in accordance with applicable GLP regulations. All relevant validity vriteria were met.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
At the start of the definitive test, two samples were taken from the freshly prepared control and test media in one set of destructive vials. After 72 hours, two further samples were taken for analysis from the remaining destructive vials for each group with algal cells. In addition, two extra vials at 1.03 and 50.0 mg/L but with no algal cells, were analysed in order to obtain information on the extent of adsorption/absorption of the test substance by the algal cells.
Vehicle:
no
Details on test solutions:
Due to the volatile nature of the test substance, the study was conducted in completely filled and sealed glass vials (nominal capacity, 65 mL). Fourteen vials were employed for each exposure group; comprising nine vials (three per occasion) for cell counts at 24, 48 and 72 hours, four vials for chemical analysis and one for water quality measurements at the end of the test (water quality measurements at the start of the test was taken from freshly prepared media).
At each concentration, an aliquot of the test substance was added to dilution medium in a volumetric flask (1 L). The contents of each flask were shaken before an aliquot of the secondary algal inoculum was added to the test medium at each concentration to provide an initial cell density of ca. 1 x 10^4 cells/mL. The test media were then used to completely fill the test vials (leaving no headspace).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokirchneriella subcapitata, Strain No. CCAP 278/4.
Axenic, uni-cellular, liquid slope cultures of algae were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Dunstaffnage Marine Laboratory, Dunbeg, Oban, Argyll, Scotland and arrived on 11 June 2013.
The liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures (100 mL) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25°C. Subsequently, appropriate volumes of these primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these cultures were incubated, as stated above, for a further three days to provide an inoculum in the log phase of growth, characterised by a cell density of 1.19 x 10^6 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not applicable
Test temperature:
Refer to Table 2.
pH:
Refer to Table 2.
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal Concentrations: 0, 1.03, 2.13, 4.70, 10.3, 22.7 and 50.0 mg/L
Measured Concentrations: 0, 0.943, 2.17, 6.71, 10.5, 23.1 and 47.2 mg/L
Details on test conditions:
A range finding test was followed by a definitive test with six test concentrations, plus an algal nutrient medium control group.
Twenty three vials were established and incubated for the control group and fourteen vials for each test group, plus an additional two vials at nominal concentrations of 1.03 and 50.0 mg/L, which contained test medium but no algal cells. Four vials from each group were allocated for chemical analysis or one for water quality measurements; these were maintained under the test conditions.
Before the start of the test, the required number of Wheaton vials (65 mL), were sterilised by autoclaving (121C for at least 15 minutes). After the addition of the inoculated test medium, each vial was sealed.
The control cultures were prepared as for the test medium except that no test substance was added and a larger volume (1700 mL) of medium was made.
Samples were taken from three sets of triplicate control and test flasks at 24, 48 and 72 hours and the cell densities measured using a Coulter Z Series Particle Count and Size Analyser; the contents of the triplicate vials on each occasion were discarded after cell counts had been conducted. The estimate of cell numbers in each sample was based on the mean of three consecutive counts, corrected for background counts of uninoculated dilution medium. The presence of any abnormal cells was also noted during screening of each test level.
Reference substance (positive control):
yes
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
9.25 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL: 8.01, 10.6)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
22.31 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL (21.6, 23.0)
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
6.71 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.17 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 95% CL (3.32, 5.78)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
11.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 95% CL (9.92, 12.5)
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
6.71 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.17 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Results with reference substance (positive control):
The results of the most recent laboratory reference test using potassium dichromate indicated that its 72-hour EbC50 to Pseudokirchneriella subcapitata was 0.73 mg/L; this was within the range typically obtained in this laboratory (0.3 to 1 mg/L).
Reported statistics and error estimates:
The data were compiled in an Excel spreadsheet and analysed using SAS 9.1 (SAS Institute 2002), Curve Fit v1.10, using the mean measured concentrations.
In order to estimate the concentration at which 50% inhibition of growth occurred (EC50), a sigmoidal curve was fitted to growth rate. 0% inhibition was defined as the control mean and 100% inhibition was defined as no growth. The minimum of the curve (for infinite concentration) was bounded between -1000 and 0 for growth rate.
Yield was calculated for each test vessel as the final cell density (after 72 hours) minus the presumed initial cell density of 1 x 104 cells/mL. A mean yield value for each test concentration was calculated and the percentage inhibition was determined.
All 95% confidence intervals for EC50 were calculated using the likelihood ratio method (Donaldson and Schnabel, 1985). The EC10 (with 95% confidence interval) was also estimated by reparameterising the above formulae.
For yield and growth rate, Williams’ test (1971, 1972) was also used to compare each treated group with the control group unless there was evidence of a non-monotonic dose-response relationship, in which case Dunnett’s test (1955, 1964) was used.
The mean coefficient of variation (CoV) for section by section daily growth rates in the control cultures was 47.0 during the definitive test and the CoV for the average specific growth rates of the control culture was 1.23 during the 72 hour exposure period.

Table 1. Measured Concentrations

Measured Allylamine Concentrations (mg/L)

 

   Nominal Concentration (mg/L)

 0 hours  % Nominal  72 hours  % Nominal  % t0  Overall Mean *
 Control  nd  -  nd  -  -  nd
 1.03  1.04  101  0.845  82  81  0.943
 1.03A  -  -  0.960  93  -  -
 2.13  2.13  100  2.21  104  104  2.17
 4.70  6.85  146  6.56  140  96  6.71
 10.3  11.0  107  10.0  97  91 10.5 
 22.7  23.6  104  22.6  100  96  23.1
 50.0  47.5  95  46.8  94  99  47.2
 50.0A  -  -  47.5  95  -  -

nd - None detected (less than the limit of detection = 0.04 mg/L)

% t0 - Measured concentration expressed as a percentage of the nominal concentration (calculated using unrounded values but expressed to 3 significant figures).

A - Culture medium incubated under test conditions without algal cells.

%N = Measured concentration expressed as a percentage of the nominal concentration (calculated using unrounded value)

* - Arithmetic mean

Table 2. Environmental Parameters

Concentration (mg/L) Temperature °C pH
Nominal   Measured  0 h  72 h  0 h  72 h
 Control  nd  23.2  22.9  7.70  9.60
 1.03  0.943  21.1  22.9  7.81  9.60
 2.13  2.17  21.1  22.8 7.75   9.56
 4.70  6.71  21.2  22.8  7.91  9.47
 10.3  10.5  21.2  22.8  7.88  9.34
 22.7  23.1  21.2  22.9  8.36 8.86 
 50.0  47.2  21.2  22.8  8.99  9.05

nd - None detected (less than the limit of detection of 0.04 mg/L)

See attached illustration for inhibition og growth data.

Validity criteria fulfilled:
yes
Conclusions:
After 72 hours of exposure to allylamine, the ErC50 and EyC50 were 22.3 and 11.2 mg/L, respectively based on the mean measured concentrations.
The no observed effect concentration (NOEC) for growth rate and yield was 2.17 mg/L (measured) and the lowest observed effect concentration (LOEC) was 6.71 mg/L.
Executive summary:

The effect of allylamine on the growth of the unicellular green alga Pseudokirchneriella subcapitata was assessed under non-axenic conditions. Due to the volatile nature of the test material the test was conducted in sealed vessels with no headspace. The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, Annex IV to Commission Regulation (EC) No 761/2009 Part C, Method 3 “Freshwater Algae and Cyanobacteria, Growth Inhibition Test” and Procedure 201 of the “Guidelines for Testing of Chemicals” of the Organisation for Economic Co-operation and Development: Freshwater Alga and Cyanobacteria, Growth Inhibition Test” (2006). Replicate algal cultures, with an initial cell density of ca. 1 x 104/mL, were exposed to allylamine dispersed in algal nutrient medium at nominal concentrations of 1.03, 2.13, 4.70, 10.3, 22.7 and 50.0 mg/L. At each concentration, the test medium was prepared by the direct addition of the test substance to dilution medium (OECD medium). The cultures were incubated in an orbital incubator under continuous illumination at temperatures ranging from 22.2 to 23.6C for 72 hours. The intended exposure concentrations of allylamine were adequately achieved (between 82 and 107% of their nominal values) and maintained during the test (between 81 and 104% of their starting values) except at 4.70 mg/L, which was anomalously high (between 140 and 146% of its nominal value). Based on an arithmetic mean, the overall measured concentrations of allylamine were 0.943, 2.17, 6.71, 10.5, 23.1 and 47.2 mg/L, and these values have been used in the calculation of test results. Cell numbers were counted daily to monitor growth; the following values were derived from the data:

 Prameter

 Mean Measured Concentration (mg/L)

Average specific growth rate (0 -72h)

EC10

EC50

LOEC

NOEC

9.25 [8.01, 10.6]

22.3 [21.6, 23.0]

6.71

2.17 

Yield (72 hours)

EC10

EC50

LOEC

NOEC

4.50 [3.32, 5.78]

11.2 [9.92, 12.5]

6.71

2.17 

[ ] 95% confidence limits

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
This study was not conducted in accordance with international guidelines, or in accordance with GLP.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
not specified
Test organisms (species):
Chlorella pyrenoidosa
Details on test organisms:
Species: Chlorella pyrenuidosa
Age/Stage: Log phase of growth
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
25 °C
Nominal and measured concentrations:
Nominal and measured concentrations tested were not reported.
Details on test conditions:
The test was inoculated at 10^4 cells per mL.
Test vessel volume: 150 mL
Reference substance (positive control):
not specified
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Validity criteria fulfilled:
not specified
Conclusions:
The 48h NOEC of allylamine to the freshwater algae Chlorella pyrenoidosa was 16 mg/L.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
This study was not conducted in accordance with international guidelines, or in accordance with GLP.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
not specified
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Selenastrum capricornutum
Age/Stage: Logarithmic stage of growth
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
26 °C
Nominal and measured concentrations:
Nominal and measured concentrations not reported.
Details on test conditions:
The test was inoculated at 5 x 10^4 cells per mL.
Test vessel volume: 150 mL
Reference substance (positive control):
not specified
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Validity criteria fulfilled:
not specified
Conclusions:
The 96h NOEC of allylamine to the freshwater algae Selenastrum capricornutum was 13 mg/L.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
This study was not conducted in accordance with international guidelines, or in accordance with GLP.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
not specified
Test organisms (species):
Scenedesmus pannonicus
Details on test organisms:
Species: Scenedesmus pannonicus
Age/Stage: Logarithmic stage of growth
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
25 °C
Nominal and measured concentrations:
Nominal and measured concentrations not reported.
Details on test conditions:
The test was inoculated at 10^4 cells per mL.
Test vessel volume: 150 mL
Reference substance (positive control):
not specified
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
17 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Validity criteria fulfilled:
not specified
Conclusions:
The 48h NOEC of allylamine to the freshwater algae Scenedesmus pannonicus was 17 mg/L.

Description of key information

72h EC50 (P. subcapitata) = 22.31 mg/L, 72h EC10 (P. subcapitata) = 9.25 mg/L; OECD 201; Allen (2013);

48h NOEC (C. pyroenoidosa) = 16 mg/L; similar to OECD 201; Canton and Adema (1978);

48h NOEC (S. pannonicus) = 17 mg/L; similar to OECD 201; Canton and Adema (1978);

96h NOEC (S. capricornutum) = 13 mg/L; similar to OECD 201; Canton and Adema (1978).

Key value for chemical safety assessment

EC50 for freshwater algae:
22.31 mg/L
EC10 or NOEC for freshwater algae:
9.25 mg/L

Additional information

A single key study (Allen, 2013) is submitted for toxicity to aquatic algae. The study was conducted in accordance with OECD 201, and was conducted in accordance with GLP. All relevant validity criteria were met in this study.