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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:
supporting study
Study period:
from 1996-07-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reference:
Composition 0
Qualifier:
according to
Guideline:
EPA OTS 797.1300 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes
Test material information:
Composition 1
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: test solutions were prepared by serial dilution of the stock solution

- Controls: control vessels contained 61 mg/L NaCl
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM

- Source: in house

- Age at study initiation (mean and range, SD): <24 h

- Feeding during test: not reported


ACCLIMATION

- Acclimation period: in house breeding

- Acclimation conditions (same as test or not): culture conditions were the same as test conditions.

- Type and amount of food: Ankistrodesmus convolutus and Nitzschia frustulum

- Feeding frequency: 4 times per week
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
Temperature ranged from 19.8-20.9 °C
pH:
pH ranged from 7.0-7.8 
Dissolved oxygen:
dissolved oxygen ranged from 8.2-8.7 mg/l i.e. >92.1% saturation
Nominal and measured concentrations:
Nominal concentrations of 61 mg/l NaCl (control), 62.5, 125, 250, 500 and 1000 mg/l QUAT 188
Nominal concentrations 61 mg/l NaCl (control), 125, 250, 500, 1000 mg/L QUAT 188 diol
Details on test conditions:
TEST SYSTEM

- Test vessel: cups

- Type (delete if not applicable): loosely covered

- Material, size, headspace, fill volume: 250 ml plastic cups filled with 200 ml test solution volume

- No. of organisms per vessel: 10

- No. of vessels per concentration (replicates): 2

- No. of vessels per control (replicates): 2


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: Lake Huron water, obtained from upper Saginaw Bay, filtered, autoclaved and pH adjusted.

- Alkalinity: hardness, 170 mg/L as CaCO3

- Culture medium different from test medium:

- Intervals of water quality measurement: Dissolved oxygen, pH and temperature were recorded in each test vessel daily.

OTHER TEST CONDITIONS

- Photoperiod: 16h light, 8 h dark cycle

- Light intensity: 1878 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobility, every 24 h

TEST CONCENTRATIONS

- Spacing factor for test concentrations: 2 (QUAT188)

- Range finding study

- Test concentrations: 0, 100 and 1000 mg/L

- Results used to determine the conditions for the definitive study: 100% mortality at 1000 mg/L (QUAT 188), 20% mortality at 1000 mg/L (QUAT 188 diol)
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
62.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobility
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
262 mg/L
Nominal / measured:
not specified
Basis for effect:
other: immobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
164 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: affected
Reported statistics and error estimates:
A computer program with probit analysis, moving average angle analysis and binomial probability/non-linear interpolation  was used to calculate the LC50/EC50 and corresponding 95 % confidence intervals. The order of preference used to select the method for reporting LC50/EC50 was: probit analysis, moving average and binomial/non-linear interpolation.  The computer program attempted to process the data using each of the three methods. However, the appropriateness of a given method was determined in the program by the concentration-response data e.g. the number of concentrations resulting in mortality between 0 and 100 %.

The 48h LC50 and EC50 for QUAT 188 were 262 mg/l (95% CI 125-500 mg/l) and 164 mg/l (95% CI 125-250 mg/l),
respectively. Effects (immobility and/or mortality) were noted in all test concentrations down to 125 mg/l.
Therefore, the NOEC was 62.5 mg/l.

The 48h LC50 and EC 50 for QUAT 188 diol were 830 (95% CI 500-1000 mg/l) and 707 (95% CI 500-1000 mg/l), respectively.
Compound-related effects (immobility and/or mortality) were noted in all test concentrations down to 1000 mg/l. Spurious
immobility was noted in the 125 and 250 mg/l dose level.
These effects were not dose responsive and did not appear to be compound-related and were not used in the determination
of a NOEC. Therefore, the NOEC was 500 mg/l.

The 48h LC50 and EC50 for QUAT 188 epoxide were 17.2 mg/l (95% CI 12.5-25.0 mg/l)and 16.4 mg/l (95% CI 12.5-25.0
mg/l), respectively. Coumpound-related effects (immobility and/or mortality) were noted in all test concentrations down
to 12.5 mg/l. Therefore, the NOEC was 6.3 mg/l.

The results reported as LC50 based on mortality should in fact be expressed as EC50, since it is hard to determine the definite mortality of small organisms such as D. magna with the naked eye. Therefore in the conclusions the LC50 will be used as EC50.

Validity criteria fulfilled:
yes
Conclusions:
A reliable 48 h EC50 value of 262 mg/L has been determined for the effects of QUAT188 on the immobility of Daphnia magna.
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes
Test material information:
Composition 1
Analytical monitoring:
no
Test organisms (species):
Daphnia magna
Test type:
static
Water media type:
freshwater
Total exposure duration:
48 h
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
1 000 - 3 200 mg/L
Nominal / measured:
nominal
Basis for effect:
mobility

EC50 was found to be > 1000 mg/l but < 3200 mg/l. At the 1000 mg/l all animals were mobile after 24 h, but at 3200 mg/l all animals were immobile after 24 h. At 1800 mg/l 11 animals of 20 were immobile and the rest 9 were slightly slower than to that of the control animals and swam somewhat closer to the bottom of the test vessels, so the EC50 was
somewhere around 1800 mg/l.

The slope of the concentration-effect curve could not be calculated.

Conclusions:
Two concentrations higher than 1000 mg of test substance per 1 l of dilution water were tested (in deviation from the protocol). There were no chemical analysis of the test substance in the test solutions. Therefore the test is ranked to be valid with restrictions; only nominal concentrations were used and no chemical analysis.
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reference:
Composition 0
GLP compliance:
yes
Test material information:
Composition 1
Test organisms (species):
Daphnia magna
Test type:
static
Water media type:
freshwater
Total exposure duration:
48 h
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Basis for effect:
other: immobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
830 mg/L
Nominal / measured:
nominal
Basis for effect:
other: immobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
707 mg/L
Nominal / measured:
nominal
Basis for effect:
mobility

The 48h EC50 (immobility, repotred as LC50 in the report) and EC50 (mobility) for QUAT 188 diol were 830 mg/l (95% CI 500-1000 mg/l) and 707 (95% CI 500-1000 mg/l), respectively.

Compound-related effects (immobility and/or mortality) were noted in all test concentrations down to 1000 mg/l. Spurious immobility was noted in the 125 and 250 mg/l dose level.
These effects were not dose responsive and did not appear to be compound-related and were not used in the determination of a NOEC. Therefore, the NOEC was 500 mg/l.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Justification for type of information:
Read-across information from the Diol is included in the present dossier as it is the main degradation product taken into account in the EU Risk Assessment Report on CHPTAC (2008).
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
1 000 - 3 200 mg/L
Nominal / measured:
nominal
Basis for effect:
mobility

EC50 was found to be > 1000 mg/l but < 3200 mg/l. At the 1000 mg/l all animals were mobile after 24 h, but at 3200 mg/l all animals were immobile after 24 h. At 1800 mg/l 11 animals of 20 were immobile and the rest 9 were slightly slower than to that of the control animals and swam somewhat closer to the bottom of the test vessels, so the EC50 was
somewhere around 1800 mg/l.

The slope of the concentration-effect curve could not be calculated.

Conclusions:
Two concentrations higher than 1000 mg of test substance per 1 l of dilution water were tested (in deviation from the protocol). There were no chemical analysis of the test substance in the test solutions. Therefore the test is ranked to be valid with restrictions; only nominal concentrations were used and no chemical analysis.

This information is used in a read-across approach in the assessment of the target substance.
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Justification for type of information:
Read-across information from the Diol is included in the present dossier as it is the main degradation product taken into account in the EU Risk Assessment Report on CHPTAC (2008).
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Test organisms (species):
Daphnia magna
Test type:
static
Water media type:
freshwater
Total exposure duration:
48 h
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Basis for effect:
other: immobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
830 mg/L
Nominal / measured:
nominal
Basis for effect:
other: immobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
707 mg/L
Nominal / measured:
nominal
Basis for effect:
mobility

The 48h EC50 (immobility, repotred as LC50 in the report) and EC50 (mobility) for QUAT 188 diol were 830 mg/l (95% CI 500-1000 mg/l) and 707 (95% CI 500-1000 mg/l), respectively.

Compound-related effects (immobility and/or mortality) were noted in all test concentrations down to 1000 mg/l. Spurious immobility was noted in the 125 and 250 mg/l dose level.
These effects were not dose responsive and did not appear to be compound-related and were not used in the determination of a NOEC. Therefore, the NOEC was 500 mg/l.

Conclusions:
It was stated that the study was conducted in compliance with the GLP with exception that chemical analysis of the test substance was not performed and therefore the test concentrations were based on nominal concentrations. The stability of the test material was determined by the sponsor prior to testing. However, no data of the stability test of test solution was given. Therefore the test is ranked to be valid with restrictions; only nominal concentrations were used and no chemical analysis.

This information is used in a read-across approach in the assessment of the target substance.
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 1988-08-11 to 1988-08-12
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
shorter exposure period, no analytical monitoring
GLP compliance:
yes
Test material information:
Composition 1
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: 10,000 mg of test substance were dissolved in 1L of dilution water, from which serial dilutions were made.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM

- Source: in house bred
- Age at study initiation (mean and range, SD): <24 h

- Method of breeding: as described in Nederlands praktijkrichtlijn NPR 6503 (1980)
- Feeding during test -
- Food type:
- Amount:
- Frequency:

ACCLIMATION
- Acclimation period: not reported, in house bred
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
Temperature of the test solutions was about 20 ± 1 °C
pH:
The pH of the test and control solutions varied between 7.3 and 8.0. 
Dissolved oxygen:
The oxygen concentrations of the test and control solutions were greater than 8.3 mg/l.
Nominal and measured concentrations:
Nominal concentrations: 32, 56, 100, 180, 560, 1000, 1800 and 3200 mg/L.
Details on test conditions:
TEST SYSTEM

- Test vessel: beakers

- Material, size, headspace, fill volume: 150 ml glass beakers filled with 100 ml test medium.

- Aeration: no

- No. of organisms per vessel: 5

- No. of vessels per concentration (replicates): 2

- No. of vessels per control (replicates): 2


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: synthetic water DSWL (as described in Nederlands praktijkrichtlijn NPR 6503 1980), prepared from ground water sourced near Linschoten.

- Alkalinity: expressed as hardness: 210 mg/L as CaCO3

- Ca/mg ratio: 2:1

- Culture medium different from test medium: no

- Intervals of water quality measurement: at 0 and 48 h

OTHER TEST CONDITIONS

- Adjustment of pH: not reported

- Photoperiod: 16 h light and 8 h dark cycle

- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobility and condition and colour compared to control after 24 h


TEST CONCENTRATIONS

- Spacing factor for test concentrations: 1.8

- Range finding study: yes

- Test concentrations: not reported

- Results used to determine the conditions for the definitive study: not reported
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
36.6 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: immobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
240.2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: immobility
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
650 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: immobility
Details on results:
- Behavioural abnormalities: It was noticed that the swimming behaviour of the animals in test solutions differed from the controls; animals swam slowly with irregular movements and closer to the bottom.  In addition, animals in test solutions were said to be paler than in controls. At a concentration of 56 mg/l (36.6 mg/l as the test substance was 65.44 %) no difference between the test solutions and controls were noticed in swimming behaviour, appearance and number of the animals. Already at a test concentration of 100 mg/l the swimming behaviour of Daphnia was different from the controls. Apparently, observations were visual, no statistical analysis was made.

- Mortality of control: 10%
Reported statistics and error estimates:
EC50 was said to be calculated by means of a parametric model developed by Kooijman.

Table 1. Number of mobile animals

 Time (h)  Concentration of test substance (mg/L)
  32  56  100  180  320  560  1000  1 800  3 200 
20  20  20 20  20  20  20  20  20  20 
24  20  20  20  19  18  13 

The results reported as whole test substance are:

24 h EC50 = 367 mg/L

24 h EC100 = 1000 mg/L

24 h NOEC = 180 mg/L

Validity criteria fulfilled:
yes
Conclusions:
A 24 h EC50 value of 240 mg/L (active ingredient, equivalent to 360 mg/L whole substance) has been determined for the effects of QUAS 188 towards the immobility of the freshwater invertebrate D. magna.

Description of key information

Four short-term studies are available for Daphnia magna. Two studies were conducted with CHPTAC and two with its main degradation product 2, 3-Dihydroxy-N,N,N-trimethylpropan-1-aminium chloride. The determined effect concentrations are in a similar range. The lowest is determined as a 48-h EC50 of 164 mg/L (nominal).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
164 mg/L

Additional information