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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 May 2013 to 28 June 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was conducted in accordance with international guidelines (OECD 202), and in compliance with the applicable GLP Regulations. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Completely filled and sealed Wheaton vials (35 mL) were used for samples. 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 48 hours, two further samples were taken for analysis from the remaining destructive vials for each group.
On each occasion, one of the samples was analysed and the other was left sealed stored at ambient temperature in case further analysis was required.
Vehicle:
no
Details on test solutions:
The method of preparation used during the definitive test was based on the results of a formulation trial and two range finding tests. Due to the volatile nature of the test substance, the study was conducted in completely filled and sealed glass vials (nominal capacity, 125 mL). The test substance also resulted in an increase in the pH of the dilution medium, so the definitive test was conducted using test media prepared with and without pH adjustment.
At each concentration, an aliquot of the test substance (13.1, 28.8, 63.5, 140 or 310 µL) was added to dilution medium (1 L) in a volumetric flask (1 L); the contents of the flask were shaken, before being used to completely fill six vials (leaving no headspace) to provide test media with nominal concentrations at 10.0, 22.0, 48.4, 106.5 or 234.3 mg/L as allylamine. No adjustment for pH was made to this batch of test media.
At each of the three highest concentrations (48.4, 106.5 and 234.3 mg/L as allylamine), a second batch of test media was prepared by adding an aliquot of test substance (63.5, 140 or 310 µL) to dilution medium (700 mL) in a volumetric flask (1 L); the pH of this media was adjusted to within 8.5 ± 0.2 using 0.1M HCl. The media were made up to volume, shaken and the pH re-measured before being used to completely fill six vials at each level, to provide the pH-adjusted test media.
Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnia magna (Straus) used in this study were cultured in-house and were obtained from a strain originating from the National Institute for Applied Chemical Research (IRCHA), France.
Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 0.5 to 0.8 litres of Elendt M4 culture medium in a temperature-controlled laboratory at nominally 20 ± 2°C. A photoperiod of 16 hours light : 8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase. The culture medium was renewed three times each week.
Cultures were fed daily with a suspension of the unicellular green algae, Pseudokirchneriella subcapitata, to provide nominally 0.1 to 0.2 mg carbon per daphnid, per day, except during the initial three days when a slightly lower ration was given. Culture conditions ensure that the stock animals reproduce by parthenogenesis.
The day before the start of the study, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by the gravid (egg-bearing) adult Daphnia were removed from the culture vessels and held in a separate holding vessel; these animals, which were less than 24 hours old, were used in the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
See Table 2.
Test temperature:
See Table 2.
pH:
See Table 2.
Dissolved oxygen:
See Table 2.
Salinity:
See Table 2.
Nominal and measured concentrations:
Nominal Concentrations: 0, 10, 22, 48.4, 106.5, 234.3 mg/L
Mean Measured Concentrations: 0, 9.61, 19.2, 43.4 (42.4), 78.7 (117), 255 (256) - Brackets denote pH adjusted media.
Further details presented in Table 1.
Reference substance (positive control):
yes
Duration:
24 h
Dose descriptor:
EC50
Remarks:
non-pH adjusted
Effect conc.:
28.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL [19.2, 43.3]
Duration:
24 h
Dose descriptor:
EC50
Remarks:
pH adjusted
Effect conc.:
194 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL [176, 256]
Key result
Duration:
48 h
Dose descriptor:
EC50
Remarks:
non-pH adjusted
Effect conc.:
28.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL [19.2, 43.4]
Key result
Duration:
48 h
Dose descriptor:
EC50
Remarks:
pH adjusted
Effect conc.:
173 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL [117, 256]
Duration:
48 h
Dose descriptor:
LOEC
Remarks:
non-pH adjusted
Effect conc.:
43.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Remarks:
pH adjusted
Effect conc.:
256 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Remarks:
non-pH adjusted
Effect conc.:
19.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Remarks:
pH adjusted
Effect conc.:
117 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
At all concentrations, the intended exposure levels of allylamine were adequately achieved (between 63 and 115% of their nominal values) and maintained during the test (between 72 and 136% of their starting values). The overall mean measured levels of allylamine were 9.61, 19.2, 43.4, 78.7 and 255 mg/L for samples non-adjusted for pH, and 42.4, 117 and 256 mg/L for samples adjusted for pH.. These values have been used in the calculation of test results.
Observations of the Daphnia in each control and test vessel were made after 24 and 48 hours. After 24 hours in non pH-adjusted media, there was 100% immobility at measured concentrations of 43.4, 78.7 and 255 mg/L. In pH adjusted media, there was 85% immobilisation at 256 mg/L, which increased to 100% at 48 hours. This implies that the pH of the test substance was responsible for observed immobilisation at nominal concentrations of 48.4 and 106.5 mg/L, with the test substance itself having a measurable impact at the highest test concentration only.
Results with reference substance (positive control):
The sensitivity of juvenile Daphnia cultured in this laboratory is periodically assessed using the reference substance potassium dichromate. The results for the most recent test performed prior to this study indicated that its 48-hour EC50 to Daphnia magna was 0.934 mg/L; this was considered comparable with data previously obtained in this laboratory.
Reported statistics and error estimates:
Statistical analysis was performed using the SAFEStat LD50 application (version 1.5), SAS 9.1.3 (SAS Institute, 2002). Test results were expressed in terms of the nominal and mean measured concentrations with and without pH adjustment.
The “no observed effect concentration” (NOEC) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% was considered to be significant.

Table 1. Measured Concentrations

 Concentration Measured Concentrations (mg/L)  Overall Mean *
 (mg/L)  0 hours  % Nominal  48 hours  % Nominal  %ti  (mg/L)
 Control  nd  -  nd  -  -  nd
 10.0  8.24  82  11.2  112  136  9.61
 22.0  17.7  80  20.8  95  118  19.2
 48.4  40.8  84  46.1  95  113  43.4
 48.4 ^  47.9  99  37.6  78  78  42.4
 106.5  92.7  87  66.8  63  72  78.7
 106.5 ^  112  105  122  115  109  117
 234.3  264  113  246  105  93  255
 234.3 ^ 260   111  253  108  97  256

nd - None detected (less than the limit of detection of 0.4 mg/L)

% ti - Mean measured concentration after 48 hours expressed as a percentage of the mean starting concentration

* Geometric mean

^ Media with pH adjustment

Table 2. Environmental Parameters

    Concentration (mg/L)     Temperature (°C)     pH     Dissolved Oxygen (% ASV)
 Nominal  Measured  0 h  48 h  0 h  48 h  0 h  48 h
 Control  nd  20.6 21.1   7.83  7.65  99  97
 10.0  9.61  20.4  21.0  8.91  8.56  99  97
 22.0  19.2  20.3  20.9  9.29  9.21  99  98
 48.4  43.4  20.3  20.7  9.77  9.43  99  96
 48.4 ^  42.4  20.3  20.7  8.43  8.35  99  96
 106.5  78.7  20.3  20.7  10.17  10.01  99  98
 106.5 ^  117  20.3  20.7  8.70  8.59  99  95
 234.3  255  20.4  20.7  10.56  10.28  100  99
 234.3 ^  256  20.3  20.8  8.68  8.62  100  96

^ - Media with pH adjustment.

nd - None detected (less than the limit of detection of 0.4 mg/L).

ASV - Air saturation value (100 % ASV = 9.09 mgO2/L at 20 °C)

The total hardness and alkalinity of the batch of Elendt M4 medium used were 236 and 49 mg/L as CaCO3, respectively.

Continuous monitoring of an additional vessel containing dilution medium = 19.8 to 20.8°C.

Table 3. Cumulative Immobilisation

    Concentration (mg/L) Cumulative Numbers of Immobile Daphnia
Nominal Mean Measured 24 hours 48 hours
     R1  R2  R3  R4  Total  %  R1  R2  R3  R4  Total  %
 Control  nd  0  0  0  0  0  0  0  0  0  0  0  0
 10.0  9.61  0  0  0  0  0  0  0  0  0  0  0  0
 22.0  19.2  0  0  0  0  0  0  0  0  0  0  0  0
 48.4  43.4  5  5  5  5  20  100  5  5  5  5  20  100
 48.4 ^  42.4  0  0  0  0  0  0  0  0  0  0  0  0
 106.5  78.7  5  5  5  5  20  100  5  5  5  5  20  100
 106.5 ^  117  0  0  0  0  0  0  0  0  0  0  0  0
234.2   255  5  5  5  5  20  100  5  5  5  5  20  100
 234.3 ^  256  5  5  3  4  17  85  5  5  5  5  20  100

nd - None detected (less than the limit of detection of 0.4 mg/L)

Rn - Replicate number.

^ - Media with pH adjustment.

Validity criteria fulfilled:
yes
Conclusions:
Based on mean measured concentrations, the 48-hour EC50 of allylamine for the immobilisation of Daphnia magna was 28.9 mg/L when performed without pH adjustment and 173 mg/L following pH adjustment of the media.
The ‘no-observed effect concentration’ (NOEC) was 19.2 mg/L without pH adjustment and 117 mg/L with pH adjustment.
These results suggested that the pH of the medium was primarily responsible for the immobilisation observed at nominal 48.4 or 106.5 mg/L as no immobilisation was observed in vessels containing media that had been pH adjusted. Immobilisation at nominal 234.3 mg/L is considered to be related to the test substance as 100% immobilisation was observed irrespective of the pH of the media.
Executive summary:

The acute toxicity of allylamine to Daphnia magna was assessed under static exposure 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 to Commission Regulation (EC) No 440/2008 Part C, Method 2 “Daphnia sp. Acute Immobilisation Test” and the OECD Guideline for Testing of Chemicals No. 202, “Daphnia Acute Immobilisation Test” (2004).

Groups of twenty Daphnia, less than 24 hours old, were exposed for 48 hours to allylamine, at nominal concentrations of 10.0, 22.0, 48.4, 107 and 234 mg/L. At each concentration, the test medium was prepared by the direct addition of the test substance to dilution medium (Elendt M4 medium). At the three highest concentrations, where the pH of the test media increased to above 9.5, additional batches of test media were prepared in which the pH was adjusted to ca. 8.5 before use.

At all concentrations, the intended exposure levels of allylamine were adequately achieved (between 63 and 115% of their nominal values) and maintained during the test (between 72 and 136% of their starting values). The overall mean measured levels of allylamine were 9.61, 19.2, 43.4, 78.7 and 255 mg/L for samples non-pH adjusted media, and 42.4, 117 and 256 mg/L for samples adjusted for pH. Both data sets have been used in the calculation of test results.

Observations of the Daphnia in each control and test vessel were made after 24 and 48 hours. After 48 hours in non pH-adjusted media, there was 100% immobility at measured concentrations of 43.4, 78.7 and 255 mg/L. In pH adjusted media, 100% immobilisation was only observed at 256 mg/L at 48 hours. This implies that it was the pH of the test media that was primarily responsible for the immobilisation observed and that the test substance itself only had a toxic effect at the highest test concentration.

Based on these findings, the following study end-points values have been determined:

  Mean Measured Concentration (mg/L)
 Parameter  Non-pH adjusted  pH adjusted
 24 -hour EC50 28.9 [19.2, 43.4]  194 [176, 256] 
 48 -hour EC50  28.9 [19.2, 43.4] 173 [117, 256] 
LOEC   43.4  256
 NOEC  19.2  117

NOEC - No observed effect concentration. LOEC - Lowest observed effect concentration. [ ] - 95% confidence intervals.

Endpoint:
short-term toxicity to aquatic invertebrates
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 according to GLP, analytical monitoring of test concentrations was not conducted, and no water quality parameters were presented.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Daphnia cucullata
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
No data
Test temperature:
The study was conducted at 19 °C.
pH:
No data
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
No data
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
>= 28 - <= 29 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: No CL published

LC50 48-hour values to Daphnia cucullata

 a*  b*  mean
 Allylamine  29  28  29

 * - Values from two seperate studies

Validity criteria fulfilled:
yes
Conclusions:
The 48-hour EC50 of allylamine to D. cucullata was 29 mg/L.
Endpoint:
short-term toxicity to aquatic invertebrates
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 according to GLP, no analytical monitoring of test concentrations was conducted and no water quality parameters are presented.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
Standard culturing techniques were used, and animals used in the toxicity test were <24 hours old at test iniation.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
No data.
Test temperature:
The study was conducted at 19 °C.
pH:
No data.
Dissolved oxygen:
No data.
Salinity:
No data.
Nominal and measured concentrations:
No data.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
>= 30 - <= 48 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: No CL published

48-hour LC50 values (mg/L) to Daphnia magna

  Laboratory 1 (exp. 1)       Laboratory 2 (exp. 1) Laboratory 2 (exp. 2)   
   a  b  mean  a  b  mean  a  b  mean
 LC50  32  30  31  42  43  43  48  46  47
Validity criteria fulfilled:
yes
Conclusions:
The 48-hour EC50 of allylamine to Daphnia magna was estimated as 30 mg/L.
Endpoint:
short-term toxicity to aquatic invertebrates
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 according to GLP, analytical monitoring of test concentrations was not conducted, and no water quality parameters were presented.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Daphnia pulex
Details on test organisms:
D. pulex were cultured using standard methods. Neonates were <24 hours old at study initiation.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
No data
Test temperature:
The study was conducted at 19 °C
pH:
No data
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
No data
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
>= 32 - <= 36 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: No CL published

LC50 48 -hour values (mg/L) to Daphnia pulex

   a  b  mean
 EC50  32  36  34
Validity criteria fulfilled:
yes
Conclusions:
The 48-hour EC50 of allylamine to D. pulex was 32 mg/L.

Description of key information

48h EC50 (Daphnia magna) = 28.9 mg/L; OECD 202; Allen (2013).

48h EC50 (Daphnia magna) = 30 mg/L; similar to OECD 202; Canton and Adema (1978).

48h EC50 (Daphnia pulex) = 32 mg/L; similar to OECD 202; Canton and Adema (1978).

48h EC50 (Daphnia cucullata) = 29 mg/L; similar to OECD 202; Canton and Adema (1978).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
28.9 mg/L

Additional information

A single key study (Allen, 2013) is submitted for acute toxicity to fish. The study was conducted in accordance with OECD 202, and was conducted in accordance with GLP. All relevant validity criteria were met in this study.