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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:
9/3/2015 to 6/5/2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg/L
- Sample storage conditions before analysis: Frozen
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain: no data
- Source: no data
- Age at study initiation (mean and range, SD): neonates, <24h
- Weight at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): no data
- Feeding during test: none
- Method of breeding: In Elendt M7 medium, at approximately 20oC, 16:8 h light/dark cycle, feeding with green algae (Desmodesmus subspicatus) and Tetramin® flake food suspension
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
no data
Test temperature:
21oC at 48 hours
pH:
6.8 after 48 hours
Dissolved oxygen:
8.8 mg/L, >60% saturation after 48 hours
Salinity:
no data
Nominal and measured concentrations:
Nominal: 100 mg/L
Measured: 0 hours: 88.5 mg/L; 48 hours: not measured
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass jar
- Material, size, headspace, fill volume: glass, 250 mL, containing 200 mL test medium
- Aeration: None
- Renewal rate of test solution (frequency/flow rate): None - static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO medium)
- Total organic carbon: No data
- Particulate matter: No data
- Metals: No data
- Pesticides: No data
- Chlorine: No data
- Alkalinity: No data
- Ca/mg ratio:No data
- Conductivity: No data
- Culture medium different from test medium: Elendt M7
- Intervals of water quality measurement: No data

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: 16:8h (light:dark)
- Light intensity: No data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : EC0 (mobility)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: none - limit test
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Key result
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.

Immobilisation forDaphnia magna (initial population 20)

24-hour

48-hour

Absolute

0

0

Cumulative

0

0

Cumulative (%)

0

0

Validity criteria fulfilled:
yes
Conclusions:
The substance had a 24 and 48 hr EC50 to Daphnia of >100 mg/L (nominal). The NOEC was 100 mg/L
Executive summary:

The 48–hour-acute toxicity of SPE1415 to Daphnia magna was studied under static conditions. The study was conducted in accordance with EU method C.2 and OECD guideline 202, in compliance with GLP. 

Daphnids were exposed to a control and limit test concentration of 100 mg/L (nominal) for 48 hours.  Analytical measurements at 0 hour confirmed that the test concentration was maintained within ± 20% of nominal. Immobilization and sub-lethal effects were observed daily.  The test results are expressed in terms of nominal concentrations. The 24 and 48–hour EC50values were 100 mg/L.  The 48–hour NOEC based on immobilization was 100 mg/L.  There were no sub-lethal effects observed.

 

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to an appropriate national standard method but full details are not available. It was not conducted under GLP and no analytical monitoring was carried out.
Qualifier:
according to
Guideline:
other: In house protocol: MIC Environmental Assessment Method for Conducting Acute Toxicity Tests with Daphnia magna (see note below)
Deviations:
no
Qualifier:
according to
Guideline:
other: US EPA (1975) Methods for acute toxicity tests with fish, macroinvertebrates and amphibians. EPA 660/3-75-009.
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
not applicable
Principles of method if other than guideline:
The In house protocol references Grueber and Adams (1980)
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: For each test concentration the appropriate amount of the test compound was pipetted into 1L of dilution water and shaken vigorously for 1 min. The solution was then divided into 2 200 ml aliquots in triplicate beakers to provide the appropriate replication. The remaining 400 ml were used for 0 h DO, pH, alkalinity and hardness determinations.

- Controls: Just dilution water.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM

- Common name: water flea

- Source:  The daphnids used in the test were obtained from a laboratory culture at the MIC laboratory.

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

- Method of breeding: according to lab SOP

- Feeding during test: no


ACCLIMATION

- Acclimation period: not reported

- Acclimation conditions (same as test or not): not reported

- Type and amount of food: Daphnia were fed a mixture of Purina trout chow and alfalfa daily.

- Health during acclimation (any mortality observed): not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
The alkalinity ranged from 236 to 304 mg/L and 250 to 300 mg/L in the low and high test concentration vessels respectively.
Test temperature:
Average temperature of 21.2 degrees C.
pH:
The pH in the control medium ranged between 7.2-8.4. The pH in the test media ranged between 2.0 and 8.2.
Dissolved oxygen:
The dissolved concentrations for the treatments were not reported due to measurement interference from the Dequest products. However, DO on the control water ranged from 6.2-9.4 mg/L. DO was measured by the Winkler titriation method.
Nominal and measured concentrations:
Nominal test concentrations were 125, 250, 500, 1000 and 2000 mg/l as active acid
Details on test conditions:
TEST SYSTEM

- Test vessel: beaker

- Material, size, headspace, fill volume: 250 ml beakers containing 200 ml of test medium. 

- Aeration: no

- No. of organisms per vessel: 10

- No. of vessels per concentration (replicates): 3

- No. of vessels per control (replicates): 3



TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: well water from St. Peters, Missouri.

- Alkalinity: 303 mg/L CaCO3, hardness 297 mg/L CaCO3

- Culture medium different from test medium: not reported

- Intervals of water quality measurement:  DO, pH, alkalinity, hardness and temperature of test solutions were monitored at the initiation (control only) and termination of the test in the high, middle and low concentrations. 


OTHER TEST CONDITIONS

- Adjustment of pH: no

- Photoperiod: not reported

- Light intensity: not reported


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobility at 0, 24 and 48 h.


TEST CONCENTRATIONS

- Spacing factor for test concentrations: 2

- Range finding study

- Test concentrations: not reported

- Results used to determine the conditions for the definitive study: EC50 833 mg/L
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
125 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: immobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
297 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: immobility
Remarks on result:
other: 95% CL 262-342
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
545 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: immobility
Details on results:
- Mortality of control: 0%
Reported statistics and error estimates:
EC50 values and 95% confidence intervals calculated using an LC50 computer programme developed by Stephen et al (1978).   

Table 1. Summary of findings (average).

 Nominal concentrations (mg/L)     Percent immobilisation 
24 h 48 h 
Control 
125  6.7 
250  13.3 
500  40  60 
1000  100  100 
2000  100  100 

Result expressed as nominal concentration. Properties of the test substance and evidence from other studies (where concentrations were 

measured) indicate that nominal and measured concentrations are likely to be in good agreement.

The pH data suggest that the acidity of the higher concentration test solutions is likely to have contributed to the effects observed in the test.


Validity criteria fulfilled:
no
Remarks:
pH very low at the highest concentrations tested. However the reported EC50 value is high enough to determine toxicity that the test substance is of low intrinsic toxicity.
Conclusions:
A 48 h EC50 value of 297 mg active acid/L has been determined for the effects of ATMP acid on the immobility of the freshwater flea D. magna. The results should be treated with caution since the authors report that the mortalities seen are an effect of the pH of the test medium, rather than a reflection of true toxicity.
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to an appropriate national standard test method. It was not compliant with GLP and no analytical monitoring was carried out. No water quality parameters have been reported, except temperature.
Qualifier:
according to
Guideline:
other: Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and Amphibians, USEPA, 1975.
Deviations:
not specified
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: a 500 ml stock solution was prepared by manual dispersion for each concentration. The solution was divided in triplicates beakers to provide replicate exposure treatments. Animals were assigned to each test beaker within 30 minutes after the compound was added.

- Controls: dilution water
Test organisms (species):
other: Chironomus tentans (aquatic insect)
Details on test organisms:
TEST ORGANISM

- Common name: midge

- Source: Midge larvae were obtained from in-house laboratory cultures at Bionomic Laboratory.

- Method of breeding: not reported

- Feeding during test: no



ACCLIMATION

- Acclimation period: not reported

- Acclimation conditions (same as test or not): same dilution water used in test

- Type and amount of food: not reported

- Health during acclimation (any mortality observed): not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
21 degrees C +/- 1
Nominal and measured concentrations:
Nominal test concentrations were 25298, 18070, 13550, 10119, and 7589 mg/l as active acid.
Details on test conditions:
TEST SYSTEM

- Test vessel: beakers. The substrate consisted of white paper towel, homogenised in water with a Waring blender. The larvae used this for construction of dwelling tubes.

- Material, size, headspace, fill volume: 250 ml vessels containing 166 ml of test solution.

- Aeration: no

- Renewal rate of test solution (frequency/flow rate): not reported

- No. of organisms per vessel: 5

- No. of vessels per concentration (replicates): 3

- No. of vessels per control (replicates): 3


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: well water, filtered through polyester fiber and sterilised with an ultraviolet steriliser to remove any micro-organisms prior to use in tests.

- Culture medium different from test medium: no

- Intervals of water quality measurement: not reported

OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: not reported
- Light intensity: not reported


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


TEST CONCENTRATIONS

- Spacing factor for test concentrations: approx. 1.35

- Range finding study: not reported
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
7 589 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
9 910 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: (7200 - 13500)
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
15 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Details on results:
- Mortality of control: 0%

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: an amber colour which appeared to be proportional to the concentration was observed.

- Effect concentrations exceeding solubility of substance in test medium: no, the water solubility of the substance is 500 g/L
Reported statistics and error estimates:
 LC50 values and 95% confidence intervals determined by least squares regression analysis of concentrations expressed as logarithms and percentage mortalities expressed as probits

Result expressed as nominal concentration. Properties of the test substance and evidence from other studies (where
concentrations were measured) indicate that nominal and measured concentrations are likely to be in good agreement.

Validity criteria fulfilled:
yes
Remarks:
for control mortality, water quality parameters not reported
Conclusions:
A reliable 48 h EC50 value of 7589 mg active acid/l has been determined for the effects of DTPMP acid on the mortality of the freshwater insect Chironomus tentans. The organisms is normally used for sediment tests, however since this is an aqueous exposure, the study has been considered in the freshwater aquatic assessment. No water quality data has been presented. Since there were no mortality in the controls and the pattern of mortality appeared regular it can be safely assumed that water quality parameters were within the norm.
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study
Qualifier:
according to
Guideline:
other: Proposed ISO Guideline and PARCOM protocol
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: direct dispersion into a stock solution

- Controls: only dilution water
Test organisms (species):
other: Acartia tonsa
Details on test organisms:
TEST ORGANISM

- Source: laboratory cultures.

- Age at study initiation (mean and range, SD): 6-8 days old

- Method of breeding: cultured unter the standard conditions according to the relevant Standard Operating Procedure

- Feeding during test: no


ACCLIMATION

- Acclimation period: not reported
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
The test was carried out at 20 degrees C
pH:
The pH of the control and all test concentrations ranged between 7.2 and 8.0.
Dissolved oxygen:
greater than or equal to 7.1 mg/l
Salinity:
ca. 28 parts per thousand
Nominal and measured concentrations:
Nominal test concentrations were 56, 100, 180, 320 and 560 mg/l.
Details on test conditions:
TEST SYSTEM

- Test vessel: scintillation vial.

- Type (delete if not applicable): covered with a perspex plate

- Material, size, headspace, fill volume: 10 ml of test medium in a vial

- No. of organisms per vessel: Each vial contained one copepodite

- No. of vessels per concentration (replicates): 10 vials per treatment. 

- No. of vessels per control (replicates): 10 vials per treatment. 


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: Natural seawater passed through sand filter, a carbon filter and then a 5um membrane filter before use. Diluted with distilled water.

- Culture medium different from test medium: no

- Intervals of water quality measurement: The pH and oxygen concentrations were measured in control and test solutions at the beginning and the end of the test.


OTHER TEST CONDITIONS

- Adjustment of pH: not reported

- Photoperiod: 16 hour light-8 hour dark cycle. And two transition periods of 0.5 of twilight.

- Light intensity: not reported


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


TEST CONCENTRATIONS

- Spacing factor for test concentrations: 1.8

- Range finding study: yes, details not reported
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
140 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 252 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Reported statistics and error estimates:
NOEC determined as highest test concentration in which at least 90% of test organisms survived. LC50 calculation not required as 50% mortality was not achieved.

Result expressed as nominal concentration. Properties of the test substance and evidence from other studies (where
concentrations were measured) indicate that nominal and measured concentrations are likely to be in good agreement.


Table 1. Mortality of Acartia tonsa during the 48 h exposure to DTPMP xNa.

 Time (h)  Nominal concentrations (mg/l)
 56  100  180  320  560
 0  0  0  0  0  0
 48  1/10 1/10   1/10  0/10  1/10  1/10


24 hr LC50: >1000 mg/l.
NOEC based on swimming behaviour/mobility.

Validity criteria fulfilled:
yes
Conclusions:
A reliable 48 h EC50 value of >252 mg active acid/l has been determined for the effects of DTPMP-xNa on the mobility of the marine copepod Acartia tonsa.

Description of key information

There is one reliable (Klimisch 1) short-term toxicity to aquatic invertebrates study available for the test substance, SPE1415. This is supported by three reliable (Klimisch 2) short-term toxicity studies in aquatic invertebrates conducted for the aminomethylphosphonate analogue substances, ATMP and DTPMP. In addition there is one reliable (Klimisch 2) study on another confidential analogue substance. These data are all included in the dossier.

One reliable (Klimisch 1) short-term toxicity study for SPE1415 with the freshwater crustacean Daphnia magna (<24 hours old) is available (Harris, 2015). The GLP-compliant study was conducted over 48 hours under static conditions. The EC50 was determined to be >100 mg/L, based on nominal test concentrations of SPE1415 (100% as UVCB).

One reliable (Klimisch 2) short-term acute toxicity study for ATMP with the freshwater crustacean Daphnia magna (<24 hours old) is available and follows published study guidelines (Monsanto Industrial Chemicals, 1981). The study was conducted under static conditions and the 48-hour EC50 was determined to be 297 mg/L based on nominal test concentrations as ATMP active acid (22%).

Two reliable (Klimisch 2) short-term toxicity studies for DTPMP with the aquatic invertebrates are available. The short-term toxicity of DTPMP was determined under static conditions using the midge Chironomus tentans and followed published study guidelines (EG&G, 1976). The 48-hour EC50 was determined to be 9,910 mg/L, based on nominal test concentrations as DTPMP active acid (18%). The short-term toxicity study of DTPMP with the marine copepod Acartia tonsa (6-8 days old at study initiation) is available (TNO Nutrition and Food Research Institute, 1997). The GLP-compliant study was conducted over 48-hours under static conditions. The 48-hour EC50 was determined to be >252 mg/L, based on nominal test concentrations as DTPMP-xNa active acid (25%).

Additional supporting data on another confidential analogue substance is discussed in Additional Information.

The short-term toxicity in aquatic invertebrates of SPE1415 has been documented within this dossier.  Reliable data exists for SPE1415 and is supported by data on aminomethylphosphonate analogues (ATMP and DTPMP).  In a conservative approach the result from the study on SPE1415 itself is used to address the hazard endpoint in question. This study resulted in an EC50 of >100 mg/L and this value will be taken as the short-term toxicity in aquatic invertebrates endpoint for SPE1415.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information