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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
exposure-related information
Qualifier:
according to guideline
Guideline:
other: EPA PB85–227049. Guidelines for deriving numerical national water quality criteria for the protection of aquatic organisms and their uses.
Qualifier:
according to guideline
Guideline:
other: EPA 600/4-91/002. Short term methods for estimating the chronic toxicity of effluents and receiving waters to aquatic organisms.
GLP compliance:
not specified
Remarks:
the present study is a public available publication.
Specific details on test material used for the study:
no data
Analytical monitoring:
yes
Details on sampling:
- Sampling method: water samples were collected weekly (from systematically selected replicates with a random starting point) in 150-mL certified clean I–Chem vials.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: NaHCO3 was added in a flow-through diluter system supplied with 250 mL of reconstituted Tongue River water every 15 min to obtain the desired experimental concentrations. The reconstituted Tongue River water generally had an alkalinity of 200 mg/L, hardness of 260 mg/L, Ca 46 mg/L, Mg 34 mg/L, Na 48 mg/L, K 0.75 mg/L, Cl 1.75 mg/L, NO 1.65 mg/L, and SO2 160 mg/L, with a pH of 8.47. Complete water replacement in each chamber occurred approximately every 4 h.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: fathead minnows
- Strain: native of Tongue and Powder River Basins of Montana and Wyoming (USA)
- Age at study initiation (mean and range, SD): eggs and less than 48h old
-source: less than 48h fish were received from Aquatic BioSystems and acclimated to increasing concentra- tions of reconstituted Tongue River (control) water for a 1-h time period.


Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
65 d
Remarks on exposure duration:
at 37 days ph (30 days exposure) half fish were sampled hystilogical examination. An additional 7d exposure test was performed with less than 48h fish for 7 days.
Hardness:
228-273 mg/L
Test temperature:
24.2-25 C
pH:
8.47-8.63
Dissolved oxygen:
5.8-6.52 mg/L
Conductivity:
651- 1180 mS/cm
Nominal and measured concentrations:
Nominal: 1st exp: 550-1400 mg NaHCO3/L. 2nd exp: 300-625 mg NaHCO3/L; 7 days exp: 300-2000 mg NaHCO3/L
Measured: The measured concentrations of mg NaHCO3/L were within10% of the nominal concentration
Details on test conditions:
TEST SYSTEM flow-trough
- Test vessel: 4-L glass exposure chambers
- fill volume: 250 mL of reconstituted Tongue River
- Type of flow-through: flow-through diluter system
- Renewal rate of test solution (frequency/flow rate): supplied with 250 mL of reconstituted Tongue River water every 15 min to obtain the desired experimental concentrations. Complete water replacement in each chamber occurred approximately every 4 h.
- No. of organisms per vessel: 70 eggs
- No. of vessels per concentration (replicates): 3 or 4
- No. of vessels per control (replicates): 3 or 4
- food: Fish were fed a ration of 10 cultured brine shrimp per fish twice per day. This ration was doubled as fish grew.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: see linked endpoint record for details


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : hatch percentage, surviva daily; lenght, weigh and hystological examinations after 30 and 60 days of exposure. The calculated hatch percentage for control and experimental concentrations was an estimate as it was not possible to count the eggs with absolute certanity due to their small size. Additional, conductivity, dissolved oxygen, and temperature were monitored daily in each replicate during the experiments. The pH, hardness, and alkalinity were measured weekly from alternate replicates so that all were sampled at least 3 times throughout the experiment.

TEST SYSTEM 7-d FHM static-renewal
- Test vessel: 1-L chambers
- fill volume: 500 mL
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates):b 3
- food: Fish were fed 200 mL of a brine shrimp suspension twice daily

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted Tongue River

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Survival was monitored daily. The experimental waters were replaced daily, and conductivity, dissolved oxygen, and temperature were monitored in a composite of the discarded replicate waters
Reference substance (positive control):
no
Key result
Duration:
37 d
Dose descriptor:
LC50
Remarks:
Experiment 1
Effect conc.:
675 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CI 486–863 mg NaHCO3/L
Key result
Duration:
30 d
Dose descriptor:
LOEC
Remarks:
Experiment 2
Effect conc.:
625 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
37 d
Dose descriptor:
LOEC
Remarks:
Experiment 1
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
30 d
Dose descriptor:
NOEC
Remarks:
Experiment 2
Effect conc.:
400 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
37 d
Dose descriptor:
other: IC20
Remarks:
Experiment 1
Effect conc.:
462 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
7 d
Dose descriptor:
LC50
Remarks:
<2dph fish exp
Effect conc.:
1 857 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CI 1507–2108mg NaHCO3/L
Duration:
7 d
Dose descriptor:
other: IC20
Remarks:
<2dph fish exp
Effect conc.:
1 190 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CI 536–643mg NaHCO3/L
Duration:
7 d
Dose descriptor:
other: IC20
Remarks:
>2dph fish exp.
Effect conc.:
499 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Remarks on result:
other: 95% CI 536–643mg NaHCO3/L
Details on results:
No significant differences were observed among treatment groups for weights and lengths. Because a t test revealed no significant difference between lengths and weights of control fish at day 37 from experiment 1 and day 30 from experiment 2, the data were combined for a pooled reference. Although no significant difference for growth was observed as a result of exposure, a significant increase in length and weight was observed at day 60 for the pooled reference when compared with those at day 30 and day 37 (14.93 mm vs 25.75 mm and 0.03 mg vs 0.18 mg), and these reference data indicate that the fish grew appropriately for the course of the experiments. An IC20 could not be calculated based on growth for FHM from experiments 1and 2.

In the flow-trough experiments, gill lesions (epithelial hypertrophy, edema, mucus, and necrosis) increased in severity with NaHCO3 concentration or the number of days of exposure. For example, at 30 d of exposure,fish from the control exhibited no gill necrosis, whereas fish from the 1100-mg NaHCO3/L exposure experi- enced moderate changes and contained focal areas of necrosis in the gills. At 60 d, large necrotic lesions were observed in the gills offish exposed to the control and 500-mg NaHCO3/L treatment but were more numerous in the 500-mg/L NaHCO3 treatment. Changes were not observed in the kidney at day 30, but at day 60 the numbers of regenerating tubules were slightly greater infish exposed to the 500-mg NaHCO3/L treatment compared with those in the controls. There were no notations about mitochondrial-rich cells during the histological examinations, and the presence of necrotic cells in the gills may have precluded observations of these types of cells. Because swelling was present, the small size of these tissues also may preclude the observation of mitochondrial-rich cells.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Significant differences for various endpoints for the 60-d FHM experiments were defined by a one-way analysis of variance, followed by either a Dunnett’s post hoc means comparison for survival or Tukey means comparison for Nþ/Kþ ATPase and whole-body ion concentrations. Data analyses were performed according to the methodology described in USEPA protocol EPA 600/4-91/002. The Toxicity Relationship Analysis Program (TRAP) was used to calculate the inhibition concentration that affects 20% of the sample population (IC20) based on mortality. The linear interpolation method was used to define IC20 based on growth or reproduction. Survival data for the 7-d experiment were analyzed with the TRAP [17] to determine the lethal concentration for 50% of the population (LC50) and the 95% confidence interval (CI). The IC20 values, calculated as mg NaHCO3/L, were based on measured rather than nominal concentrations, except for the 7-d FHM experiment. The trimmed Spearman–Karber program was used to calculate a 10-d effect concentration on 50% of the sample population (EC50).

The hatch percentage for each treatment mg NaHCO3/L was 63% for the control, 78% for 500 mg NaHCO3/L, 69% for 800 mg NaHCO3/L, 70% for 1100 mg NaHCO3/L, and 44% for 1400 mg NaHCO3/L. Although not tested statistically because these were estimates, the percentage of eggs that hatched at 1400 mg NaHCO3/L was smaller compared with the controls during experiment 1.

Table 2. Survival percentage of fathead minnows (Pimephales promelas) during chronic experiments (flow-through) completed in reconstituted Tongue River water (control)a

Fathead minnow experiment1

Fathead minnow experiment 2

Exposureb

(mg NaHCO3/L)

Survivalpercentage

0–37 d

Survival percentage

37–60 d

Exposure

(mg NaHCO3/L)

Survival percentage

0–30 d

Survival percentage

30–60 d

Control

96

96

Control

68

99

500

64*

93

300

35

100

800

33*

+c

400

57

100

1100

21*

+

625

30*

100

1400

2*

+

--d

--

--

aEgg mortality from “estimated hatch percentage” reported in the Results section is not included in the survival percentage calculations. Survival percentage is not cumulative but is reported at the end of each time period.
bNominal concentrations of NaHCO3
cSacrificed concentrations at day 37.

dNo concentration exposed or data gathered.

*p < 0.05.

Table 3. Mean survival percentage and weights at the end of a 7-d static renewal chronic exposure of fathead minnow (Pimephales promelas) completed in reconstituted Tongue River water (control)a

Exposureb

(mg NaHCO3/L)

7-d mean no. of individuals

Survival (%)

Day 7 weight (mg)

Control

10 (0)

100

0.005 (0.0005)

300

10 (0)

100

0.006 (0.001)

625

9.3 (0.6)

93

0.004*(0.0004)

800  

10 (0)

100

0.004*(0.00007)

1100

7.7*(2.3)

77

0.004*(0.0008)

1400

7.7*(1.5)

77

0.003(0.0006)

2000

4.3*(1.2)

43

0.003 (0.0009)

aStandard deviation in parentheses. Standard deviation in parentheses.

bNominal concentrations of NaHCO3; sample size=3. Nominal concentrations of NaHCO3; sample size=3.

*p < 0.05

Conclusions:
Eggs of the fish fathead minnow were exposed for 60 days to NaHCO3. Reasults showed and LC50 for mortality at 37days of 675 mg/L NaHCO3. Weight and lenght were not affected after 60 days of exposure.
Additional experiment performed with >2dph fish exposed for 7 days to NaHCO3 resulted in a LC50 for mortality of 1857 mg/L and an IC20 for changes in weight of 780 mg/L NaHCO3.
Executive summary:

A study was conducted exposing newly fertilezed eggs of the fish fathead minnow, Pimephales promelas, for 60 days in a flow-through system to a concentration range of 300-1400 mg NaHCO3/L in two separated experiments (1stexp: 550-1400 mg NaHCO3/L; 2ndexp: 300-625 mg NaHCO3/L). Half of the fish were sampled at 30-37 days after exposure for histopathological examination. Results showed overall an LC50 at 37 days of 675 mg/L NaHCO3and a NOEC of 400 mg/L NaHCO3based on mortality. Changes in weight and length were not significant different after 30-37 of exposure compared with the control. After 30 days of exposure, histopathological observations revealed an increase in gill lesions severity with increasing exposure concentration, whereas kidney changes where observed only at the end of the experiment (60 days).

In addition, 7 days static test was performed using less than 48 h fish exposed to a range concentration of 300-2000 mg NaHCO3/L. Results showed LC50 for mortality of 1857 mg/L and an IC20 for changes in weight of 780 mg NaHCO3/L.

 

Description of key information

A reliable publication (K 2) is present exposing two fish species, fathead minnow (Pimephales promelas) and white sucker (Catostomus commersoni), for 60 days to NaHCO3 at concentration range of 300-1400 and 450-1400 mg/L, respectively.

Experiment with newly fertilised eggs of fathead minnow was chosen as key study as this is a preferred OECD standard species for long-term studies. Results showed overall an LC50 at 37 days of 675 mg/L NaHCO3 and a NOEC of 400 mg/L NaHCO3 based on mortality. Changes in weight and length were not significant different after 30-37 of exposure compared with the control. After 30 days of exposure, histopathological observations revealed an increase in gill lesions severity with increasing exposure concentration, whereas kidney changes were observed only at the end of the experiment (60 days).

 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
400 mg/L

Additional information

A study was conducted exposing newly fertilised eggs of the fish white sucker, Catostomus commersoni, for 60 days in a flow-through system to a concentration range of 450-1400 mg/L NaHCO3. At the end of the experiment mortality in treated fish was not significant different of the control, however growth was affected. Results showed LOEC for changes in weight and length of 450 mg/L NaHCO3 and an IC20 for changes in weight of 348 mg/L NaHCO3.

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