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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Test organisms (species):
other: Dreissena polymorpha pallas
Details on test organisms:
TEST ORGANISM
- Common name: Zebra mussel
- Source: Collected from the lake of Salagou (Hérault)
- Age: 3-4 years
- Size: 20 - 30 mm
- Food type: Liquifray Marine


ACCLIMATION
- Acclimation period: 1 week
Test type:
other: Semistatic and dynamic
Test temperature:
17 - 25 °C
Dissolved oxygen:
Equivalent to 90%
Nominal and measured concentrations:
The tests with crude cyanuric acid and purified cyanuric acid in a semistatic medium were conducted at 500, 1000 and 2000 mg/L.
Details on test conditions:
The test was conducted in glass aquaria
Reported statistics and error estimates:
For the calculation of the LT (lethal exposure time) the experiments consisted of noting the survival time of each organism exposed to a given concentration. The logarithmic transformation of the survival times and that of the cumulative mortality percentages in probits allowed the generation of a straight line adjusted by the 'least squares' method (Bliss, 1957). From the equation of the straight line the LT10, LT50 and LT990 values were determined. The LT values were expressed in days.

The series of tests conducted with crude cyanuric acid (CCA) and purified cyanuric acid (PCA) in a semistatic medium at concentrations of 500, 1000 and 2000 mg/L allowed the calculation of the LT50and the confidence interval. Inspection of the LT50values distinguished two types of toxicity: a long term toxicity for the three concentrations of CCA and for the concentration of 500 mg/L for PCA, whose LT50values were higher at 1 month, as well as a medium term toxicity for the concentrations of 1000 and 2000 mg/L of PCA, whose LT50values were 16 and 17 days respectively.

 

The LT10 values obtained reflect the 5 times more rapid appearance of the lethal effect with PCA at 1000 and 2000 mg/L (LT10= 1 week) compared to PCA at 500 mg/L and CCA at the three concentrations tested (LT10> 5 weeks). The LT90values for the PCA at the concentration of 1000 and 2000 mg/L. (LT90~ 1 month) reflect an effectiveness that is two times better than with PCA at 500 mg/L and CCA at the three concentrations tested (LT90> 2 months).

 

The study of the physicochemical properties conducted during the course of the tests reflected an increase over time in the values of pH as well as concentrations of Ca++and (NH3 + NH4+). The pH values which are acidic at the beginning (6.2 ≤ pH ≤ 6.8), increase and stabilize after 48 h (7.5 ≤ pH ≤ 7.9.). The lower the starting pH values, the greater the increase in the Ca++concentrations. The increase n the level of ammonical nitrogen is more pronounced with CCA than with PCA. At the end of the experiment, the valves of the molluscs had been attacked or perforated in the vicinity of the apex, whereas no sign of alteration was observed in the controls. This alteration of the shells appeared earlier with PCA than with CCA; it was related to the concentrations of the two products.         

Conclusions:
Raw cynauric acid revealed to be less toxic (LC50 1000 and 2000 mg/L > 1 month) than pure cyanuric acid (LT50 1000 and 2000 mg/L ≤ 17 days). The cyanuric acid seemed to act on 2 levels: 1) by valve decalcification and 2) by deterioration of kidney function with accumulation of the product.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and each surviving test group (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on day 0 and of the expired test preparations on days 2, 5, 7, 9, 12, 14, 16, 19 and 21. Duplicate samples and samples of the freshly prepared solutions on days 2, 5, 7, 9, 12, 14, 16 and 19 were taken and stored at approximately -20°C for further analysis if necessary.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
Amounts of test material (10000 and 5000 mg) were each seperately dissolved with the aid of ultrasonication for approximately 30 minutes at approximately 70-80°C in reconstituted water and the volumes adjusted to 2 and 1 litres respectively. Solutions were heated to aid dissolution. The stock solutions were then cooled to approximately 21°C and pooled prior to filtration through a 0.2 µm filter to give the 5000 mg/l test concentration. Aliquots (20, 64, 200 and 640 ml) of the 5000 mg/l test concentration were each separately dispersed in a final volume of 2 litres of reconstituted water to give the remainder of the test concentrations of 50, 160, 500 and 1600 mg/l respectively.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: In house laboratory cultures
- Age: 1st instar
- Feeding during test: Each daphnid received approximately 5 to 10 µl of a unicellular algal culture (Chlorella sp.) daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Adult Daphnia were transferred to fresh media 3 times a week by wide bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted using stereo microscope.
Test type:
static
Water media type:
freshwater
Total exposure duration:
21 d
Hardness:
Refer to table 1
Test temperature:
Temperature was maintained at 18.7 to 20.4°C .Refer to attached document for measurements taken throughout the study
pH:
Concentration dependent differences in pH were observed throughout the tests. Refer to attached document for measurements taken throughout the study
Dissolved oxygen:
Refer to attached document for measurements taken throughout the study
Nominal and measured concentrations:
50, 160, 500, 1600 and 5000 mg/L (nominal) (equivalent to 37.8, 121, 378, 1210 and 3780 mg cyanuric acid/L).
Refer to table 2 for measured concentrations taken throughout the test.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 100 mL solution in 150 mL glass flasks
- Aeration: The diluent water only was aerated prior to use.
- Renewal rate of test solution: Test preparations were renewed 3 times per week on days 0, 2, 5, 7, 9, 12, 14, 16 and 19.
- No. of organisms per vessel: 1
- No. of vessels per concentration : 10
- No. of vessels per control: 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water
- Hardness: 250 mg/l as CaCO3
- Ca/mg ratio: CaCl2.2H2O: 11.76 g/L , MgSO4.7H2O: 4.93 g/L
- Conductivity: < 5 µS cm-1


OTHER TEST CONDITIONS
- Photoperiod: 16 h light, 8 hours dark with 20 minute dawn and dusk transition periods.
- Light intensity: 521-537 lux

EFFECT PARAMETERS MEASURED: Immobilisation and reproduction. On a daily basis the numbers of live and dead of the ‘parental’ (P1) generation, the numbers of live and dead ‘filial’ (F1) daphnia and the number of discarded unhatched eggs were counted.

Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
2 600 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks on result:
other: Equivalent to 1966 mg/l cyanuric acid
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
2 800 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: equivalent to 2117 mg/l cyanuric acid
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: mortality and reproduction
Remarks on result:
other: equivalent to 378 mg/l cyanuric acid
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
160 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: mortality and reproduction
Remarks on result:
other: equivalent to 121 mg/l cyanuric acid
Details on results:
Refer to table 3 and table 4
Reported statistics and error estimates:
The EC50 (immobilisation) values up to day 14 of the test were estimated by inspection of the data. The EC50 (immobilisation) value at day 21 was calculated by the maximum-likelihood logit method (Finney 1978) using the ToxCalc computer software package (ToxCalc 1999). 95% confidence limits could not be calculated due to the unsuitable nature of the data resulting from a flat dose response.
Logit analysis is used where two or more partial responses to exposure are shown. This method of statistical analysis was used as opposed to the probit method due to the unsuitable nature of the data for analysis by probit.
The observed mortalities in the parental (P1) generation of the 50 and 160 mg/L test groups were compared to the control group using the chi-squared statistic (Breslow and Day 1980).
The EC50 (reproduction) value and associated confidence limits after 21 days were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).
Probit analysis was used where two or more partial responses to exposure are shown.
For the estimation of the “Lowest Observed Effect Concentration” (LOEC) and the “No Observed Effect Concentration” (NOEC) the numbers of live young produced per adult over the duration of the test control and each test group were compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). Results from the control and each test group’s Daphnia length data, determined for the surviving daphnids on termination of the test, were compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). Statistical analyses were performed using the SAS computer software.

Table 1: Water hardness measurements

Day

Water hardness (mg/l as CaCO3)

Control

Highest surviving concentration

0 (fresh media)

260

244

2 (old media)

252

240

7 (fresh media)

252

134

9 (old media)

258

226

14 (fresh media)

250

126

16 (old media)

248

94

Table 2: Measured concentrations of test substance:

Day

Nominal concentration (mg/L)

Control

50

160

500

1600

5000

0

less than LOQ1

49.6

161

502

1610

4990

2

less than LOQ

49.4

160

502

1600

4940

5

less than LOQ

50.0

163

517

1650

5000

7

less than LOQ

49.2

160

494

1590

4910

9

less than LOQ

49.4

160

498

1590

4950

12

less than LOQ

49.2

161

504

1600

3810/147000*

14

less than LOQ

48.6

158

503

1600

4900

16

less than LOQ

49.2

160

498

1600

4880

19

less than LOQ

50.5

160

486

1570

4750

21

less than LOQ

4901

158

486

1580

4820

1 LOQ = Limit of quantitation

* Duplicate sample, stored frozen prior to analysis - considered as erroneious result due to analytical error

.

Table 3: Daphnia lengths of the parental generation

Nominal concentration (mg/l)

Daphnia length at day 21 (mm)

Control

Mean

Standard deviation

4.5

0.3

50

Mean

Standard deviation

4.6

0.3

160

Mean

Standard deviation

4.5

0.5

500

Mean

Standard deviation

4.5

0.3

1600

Mean

Standard deviation

4.5

0.2

5000

Mean

Standard deviation

4.1*

0.2

* Significant differences (p <0.05)

Table 4: Summary of findings following the exposure of Daphnia magna for 21 days

Nominal concnetration (mg/l)

% survival of P1

Number of live young

Number of  dead young

Number of unhatched eggs

Total

Per female (cumulative)

Total

Per female (cumulative)

Total

Per female (cumulative

Control

80

611

76

0

0

0

0

50

90

702

78

0

0

0

0

160

80

515

64

1

1

2

1

500

30

232

77

1

1

1

1

1600

50

340

68

1

1

0

0

5000

70

52

7

348

50

1

0

.

Validity criteria fulfilled:
yes
Remarks:
Control mortality: 20%, Dissolved oxygen > 7.3 mg O2/l, pH (control): 0.5, Mean number of live young surviving adult (control group): 76, Coefficient of variation for control group: 9%
Conclusions:
Exposure of Daphnia magna to monosodium salt of cyanuric acid resulted in signifciant mortalities at the test concentrations of 500,1600 and 5000 mg/l resulting in 30%, 50% and 70% mortalities by day 21 respectively, compared to an observed mortality of 20% in the control by day 21.
The 21 day EC50 (immobilisation) values, based on nominal test concentrations, for the parental Daphnia (P1) was calculated to be 2600 mg/l. 95% confidence limits could not be calculated due to the unsuitable nature of the data resulting from a flat dose response.
The 21-day EC50 (reproduction) based on nominal test concentrations was 2800 mg/l with 95% confidence limits of 1800 - 4400 mg/l.

Description of key information

A Daphnia magna reproduction study (OECD 211) performed with the Monosodium salt of cyanuric acid (equivalent to 75.6% cyanuric acid).    

Key value for chemical safety assessment

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

Additional information

A Daphnia magna reproduction study (Sewell 2007) was performed with the monosodium salt of cyanuric acid (equivalent to 75.6% cyanuric acid). Daphnia were exposed to nominal concentrations of 50, 160, 500, 1600 and 5000 mg/L (equivalent to 37.8, 121, 378, 1210 and 3780 mg cyanuric acid/L) for a period of 21 days.The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. Exposure of Daphnia magna to monosodium salt of cyanuric acid resulted in significant mortalities at the test concentrations of 500,1600 and 5000 mg/L resulting in 30%, 50% and 70% mortalities by day 21 respectively, compared to an observed mortality of 20% in the control by day 21. The NOEC was considered to be 160 mg/L (equivalent to 121 mg/L CYA) on the basis that at this concentration there were no significant mortalities (immobilisation) observed in the parental generation (P1) and that there were no significant differences between the control and the 160 mg/L test group in terms of numbers of live young per adult by day 21.