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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Feb 19, 2014 to March 26, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
Principles of method if other than guideline:
.
GLP compliance:
yes
Remarks:
according to OECD Good Laboratory Practice Standards
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 0.048, 0.095, 0.19, 0.38, and 0.75 mg a.i./L
Sampling frequency: The concentration of Colipa A050 was measured in test solution samples collected on days -1 (1 day prior to test initiation), 0, 7, 14, and 21 during the definitive test.
- Sampling method: At each sampling point, 9-mL samples were collected into appropriately labeled culture tubes. The samples were diluted to 10 mL with 2% erythorbic acid in HPLC grade water and further diluted, if necessary, with 0.2% erythorbic acid in HPLC grade water to provide final concentrations within the analytical standard concentration range (i.e., 0.0268 to 0.856 mg a.i./L).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Diluter stock solutions were prepared on a daily basis during the definitive test at a target concentration of 75 mg a.i./L. The solutions were prepared by weighing approximately 0.0605 grams of Colipa A050 (0.0600 grams adjusted for 99.24% purity) and diluting to a volume of 0.80 L, respectively, with laboratory deionized water.
- A syringe dispenser introduced 10-mL volumes of the diluter stock solution to the chemical mixing box, where it was diluted with approximately 990 mL of dilution water to generate the highest test substance treatment solution. The 10-mL volume of the diluter stock solution was added to the 990 mL of dilution water to the system further diluting the solution to prepare the four lower test substance treatment solutions and delivering the freshly prepared highest test substance treatment solution to the appropriate test chambers.
- Control: The negative control consisted of dilution water only.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: not reported
-Source: Test specimens of Daphnia magna were obtained from an in-house culture
- Culturing of daphnids: The culture was maintained on a 16-hour light:8-hour dark photoperiod with 30-minute dawn/dusk transitions periods. During the holding period, the daphnids were fed a suspension of the algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) at least once a day supplemented by a prepared artificial diet consisting of a wheat grass, salmon starter, and yeast suspension.
- Age at test begin: first instar ( <24 hours old)
- Feeding during test: yes
- Amount and Frequency: 3.0 × 10(7) cells/mL Pseudokirchneriella subcapitata and a prepared invertebrate food solution (YCT daphnid feed mixture) containing 2.3 or 2.4 g of suspended solids/mL. 3-6 mL of the algal suspension were manually added to each replicate chamber four times daily (e.g., early morning, late morning, early afternoon, and late afternoon), except on the day of test initiation (i.e., Day 0) when 3 mL was added to each replicate on just three occasions. The invertebrate food solution was manually added to each replicate chamber three times daily. The volume of the prepared invertebrate food solution offered at each feeding was 1 mL.

ACCLIMATION: Since the culturing and testing environmental parameters were equivalent (i.e., temperature, dilution water, and lighting), no acclimation period was necessary.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
142 to 148 CaCO3/L
Test temperature:
20.4 to 21.0°C
pH:
8.2 to 8.4
Dissolved oxygen:
7.4 to 8.4 mg/L (87 to 99% saturation)
Nominal and measured concentrations:
Nominal test concentrations: 0 (control), 0.048, 0.095, 0.19, 0.38, and 0.75 mg a.i./L
Mean measured test concentrations:
Details on test conditions:
TEST SYSTEM
- Test vessel: beaker
- Material: Glass beakers measuring approximately 15 cm in height and 10 cm in diameter. The depth of the test solution was maintained at approximately 12 cm. The volume of each test chamber was approximately 1 L
- Aeration: no
- Type of flow-through and Renewal rate of test solution: intermittent-flow proportional diluter system. The diluter system mixing cells delivered the dilution water control and each of the five test solutions to flow-splitting cells. Each flow-splitting cell divided each 500-mL volume four ways. This split resulted in a volume of approximately 125 mL being delivered to each test chamber with each cycle. The frequency of the 125-mL additions was maintained at a rate sufficient to provide six volume additions, which was greater than the minimum five volume additions, to each test chamber in a 24-hour period. The diluter system cycle rate was approximately 2.0 cycles/hour.
- No. of organisms per vessel: 10
- No. of vessels per concentration: 4
- No. of vessels per control : 4
- Biomass loading rate: 1 daphnid per 100 mL of dilution water

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was a moderately hard freshwater prepared by blending naturally hard well water with well water that was demineralized by reverse osmosis (RO). The well water and RO water was blended to yield a total hardness of 140 to 160 mg CaCO3/L. Prior to delivery to the test chambers, the dilution water was passed through a sediment filter and UV sterilizer The characteristics of dilution water were:
Total organic carbon: was less than 2.0 mg/L
Alkalinity: 154 to 158 CaCO3/L
Hardness: 142 to 148 mg CaCO3/L
Conductivity: 336 to 343 μS/cm
Further details are provided in “Appendix C” of study report.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Total hardness, total alkalinity, and conductivity were measured in the dilution water on days 0, 7, 14, and 21 of the definitive test. Water quality characteristics of temperature, dissolved oxygen concentration, and pH were measured in each replicate of the control and each test substance treatment at initiation and once each week following initiation.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16-hour daylight, 8-hour darkness, and 30-minute simulated dusk and dawn transition periods
- Light intensity: 392 to 506 lux

EFFECT PARAMETERS MEASURED: Observations were made daily on the number of surviving adult daphnids, occurrence of abnormalities, and production of neonates.

RANGE-FINDING STUDY: A 14-day flow-through range-finding test was initiated at nominal concentrations of 0 (control), 0.033, 0.065, 0.13, 0.25, and 0.50 mg a.i./L.
Results of range finding study: After 14 days of exposure there was 95, 100, 95, 100, 95, and 100% survival in the 0 (control), 0.033, 0.065, 0.13, 0.25, and 0.50 mg a.i./L substance treatments, respectively.
- The total number of young produced during the 14-day exposure was 653, 939, 727, 989, 581, and 845 neonates in the 0 (control), 0.033, 0.065, 0.13, 0.25, and 0.50 mg a.i./L test substance treatments, respectively.
- The mean number of young produced per living adult after 14 days of exposure were 34, 47, 38, 49, 31, and 42 neonates in the 0 (control), 0.033, 0.065, 0.13, 0.25, and 0.50 mg a.i./L test substance treatments, respectively. Based on these results, nominal concentrations of 0 (control), 0.048, 0.095, 0.19, 0.38, and 0.75 mg a.i./L were selected for the definitive test.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.674 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.674 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
immobilisation
Details on results:
- Mortality: Survival of the control daphnids was 93%. There was no statistically significant decrease of survival, as compared to the control (i.e., 93% survival) in any of the test substance treatments.

- Time to first brood: The average number of days until first brood was nine days in the control. The average number of days the first brood was observed in the test substance treatments ranged from eight to nine days.

- Mean number of young produced per parent: The mean number of young produced per parent present at test initiation in the control was 82, and ranged from 70 in the 0.335 mg a.i./L treatment to 86 in the 0.0363 mg a.i./L treatment. No other immobility or morphological abnormalities were observed in the young of any treatment. The mean number of young produced per surviving parent present at test termination in the control was 89. The mean number of young per surviving parent in the test substance treatments ranged from 83 in the 0.674 mg a.i./L treatment to 100 in the 0.0720 mg a.i./L treatment.

-Number of young per reproductive day: The number of young per reproductive day was 5.9 in the control, and ranged from 5.6 in the 0.335 and 0.674 mg a.i./L treatments to 6.3 in the 0.0363 and 0.0720 mg a.i./L treatments.

For details, refer to “Table 1” under Any other information on results incl. tables
Reported statistics and error estimates:
- All statistical analyses were performed using SAS software (version 9.3). The outlier test was conducted using RStudent test for the survival data.

- The no-observed-effect concentration (NOEC) and lowest-observed-effect concentration (LOEC) for mortality/immobility data were determined by using a one-tailed Dunnett’s test and a Fisher’s exact test with the alternate hypothesis being that the mean for the parameter was reduced in comparison to the control mean. A Hochberg adjustment was used to control the experiment-wise error rate for the Fisher’s test at the same level (p = 0.05).

- Reproduction data was analyzed with a one-way analysis of variance (ANOVA) procedure and a one-tailed Dunnett’s test with the alternate hypothesis being that the mean for the parameter was reduced in comparison to the control mean. Prior to the Dunnett’s test, a Shapiro-Wilk’s test and a Levene’s test were conducted to test for normality and homogeneity of variance, respectively, over treatments at each time point. The results from the Shapiro-Wilk’s and Levene’s tests indicated the assumptions of normality and homogeneity of variance were met for all the reproductive data.

Table 1: Study results of 21-Day Chronic Flow-Through Toxicity Test with Daphnia magna exposed to Colipa A050 (study # 68344)

Mean Measured Concentration (mg a.i./L)

Treatment % Survival

Mean Day of First Brood

Offspring/Parent at Study Start

Offspring per Surviving Parent

Offspring per Adult Reproductive Daya

0 (Control)

93

9

82

89

5.9

0.0363

90

9

86

96

6.3

0.072

85

8

85

100

6.3

0.164

85

9

79

95

6.1

0.335

80

9

70

90

5.6

0.674

97

8

80

83

5.6

aTotal number of young per adult reproductive day. Total reproductive days are the total number of adult females once they start producing young until the termination of the study, which is typically Day 21.

Validity criteria fulfilled:
yes
Conclusions:
In a 21 day chronic toxicity test to Daphnia magna, the NOEC of N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulphate was 0.674 mg a.i./L (highest tested concentration), based on mean measured concentrations. The endpoints evaluated were survival and offspring per adult.
Executive summary:

A 21 d chronic toxicity test of N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulphate to Daphnia magna was determined under flow-through conditions, following OECD Guideline 211 (Daphnia magna Reproduction Test).

Nominal test concentrations were 0 (control), 0.048, 0.095, 0.19, 0.38, and 0.75 mg a.i./L. Mean measured test concentrations <MQL (control), 0.0363, 0.0720, 0.164, 0.335, and 0.674 mg a.i./L which represented 76 to 90% of the nominal concentrations. There were 4 replicates at each test concentration and control level. Ten daphnids were added initially to each replicate test chamber. The dilution water was a moderately hard freshwater prepared by blending naturally hard well water with well water that was demineralized by reverse osmosis (RO).

Survival of the control daphnids was 93%. There was no statistically significant decrease of survival, as compared to the control (i.e., 93% survival) in any of the test substance treatments. The average number of days until first brood was nine days in the control. The average number of days the first brood was observed in the test substance treatments ranged from eight to nine days.

The mean number of young produced per parent present at test initiation in the control was 82, and ranged from 70 in the 0.335 mg a.i./L treatment to 86 in the 0.0363 mg a.i./L treatment. No other immobility or morphological abnormalities were observed in the young of any treatment. The mean number of young produced per surviving parent present at test termination in the control was 89. The mean number of young per surviving parent in the test substance treatments ranged from 83 in the 0.674 mg a.i./L treatment to 100 in the 0.0720 mg a.i./L treatment. The number of young per reproductive day was 5.9 in the control, and ranged from 5.6 in the 0.335 and 0.674 mg a.i./L treatments to 6.3 in the 0.0363 and 0.0720 mg a.i./L treatments.

All the validity criteria were met. Since, significant concentration-dependent adverse biological effects were not observed for any of the endpoints, NOEC and LOEC for all biological effects were 0.674 and >0.674, respectively, the highest concentration tested. The  EC50 (21d) as well was defined as > 0.674 mg/L.

This chronic toxicity test to daphnids is classified as acceptable, and satisfies the guideline requirements for the OECD Guideline 211 (Daphnia magna Reproduction Test).

Description of key information

In a 21 day chronic toxicity test to Daphnia magna, the NOEC of N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulphate was 0.674 mg a.i./L (highest tested concentration), based on mean measured concentrations. The endpoints evaluated were survival and offspring per adult.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.674 mg/L

Additional information

A 21 d chronic toxicity test of N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulphate toDaphnia magnawas determined under flow-through conditions, following OECD Guideline 211 (Daphnia magna Reproduction Test).

Nominal test concentrations were 0 (control), 0.048, 0.095, 0.19, 0.38, and 0.75 mg a.i./L. Mean measured test concentrations <MQL (control), 0.0363, 0.0720, 0.164, 0.335, and 0.674 mg a.i./L which represented 76 to 90% of the nominal concentrations.There were 4 replicates at each test concentration and control level. Ten daphnids were added initially to each replicate test chamber. The dilution water was a moderately hard freshwater prepared by blending naturally hard well water with well water that was demineralized by reverse osmosis (RO).

Survival of the control daphnids was 93%. There was no statistically significant decrease of survival, as compared to the control (i.e., 93% survival) in any of the test substance treatments. The average number of days until first brood was nine days in the control. The average number of days the first brood was observed in the test substance treatments ranged from eight to nine days.

The mean number of young produced per parent present at test initiation in the control was 82, and ranged from 70 in the 0.335 mg a.i./L treatment to 86 in the 0.0363 mg a.i./L treatment. No other immobility or morphological abnormalities were observed in the young of any treatment. The mean number of young produced per surviving parent present at test termination in the control was 89. The mean number of young per surviving parent in the test substance treatments ranged from 83 in the 0.674 mg a.i./L treatment to 100 in the 0.0720 mg a.i./L treatment. The number of young per reproductive day was 5.9 in the control, and ranged from 5.6 in the 0.335 and 0.674 mg a.i./L treatments to 6.3 in the 0.0363 and 0.0720 mg a.i./L treatments.

All the validity criteria were met. Since, significant concentration-dependent adverse biological effects were not observed for any of the endpoints, NOEC and LOEC for all biological effects were 0.674 and >0.674, respectively, the highest concentration tested.The  EC50 (21d) as well was defined as > 0.674 mg/L.