Salinity Tolerance Lab 3

Diamonique Harris Dr. Amanda Glazier Biology 3244 Lab June 13, 2018 Salinity Tolerance Introduction: Salinity can be a major survival determining factor in marine organisms. The level of sensitivity to changes in salinity varies from organism to organism, which is why not all organism can survive in environment where the salinity fluctuates. Organisms can be characterized into two categories for tolerance of salinity, stenohaline and euryhaline. Stenohaline, are organisms that have the inability to tolerate fluctuating salinities and are mainly found in either freshwater or saltwater environments (NOAA) such as the open ocean and the deep sea. Whereas, the euryhaline are organisms that can tolerate large changes in salinity and are more common along shorelines and in estuaries, where rainfall and river runoff can create large fluctuation in salinity. Stenohaline organisms because it requires a lot of energy to adapt to constantly changing salinities (NOAA). In order for the stenohaline organisms to maintain their salt balance there are two mechanisms that they can use, osmoregulators and osmoconformers. The process is which an organism can control their concentrations of salts of water in internal fluids despite the levels of the surroundings. Osmoregulators maintain their internal salinity through the use of salt glands, kidneys and/or gills all of which cost an extreme amount of energy to regulate their fluids. As opposed to, osmoconformes change their internal salinities with respect to the changes in their external salinity. When the organism is hypertonic to its environment, water moves into the organism due to the internal salts in the organism being greater than the external environment. On the contrary, water is lost from the organism, when the organism becomes hypotonic to the

environment, in other words, the environment becomes saltier than the internal fluid of the organism. An example of an osmoregulating organism that can survive in high salinity is the aquatic crustaceans, brine shrimp Artemia . Due to the Artemia ability to absorb and excrete salts through their gills makes them perfect osmoregulators and ability to tolerate ranges of salinity. However, the Nucella lapillus , are osmoconformers and respond to large fluctuation in salinity by closing up their shells to prevent excessive dilution/desiccation of internal fluids. The purpose of this experiment is to get an understanding of the effects that salinity changes could have on osmoregulators and osmoconformers such as Artemia and Nucella lapillus (dogwhelks). In order to get a clearer understanding of osmoregulators and osmoconformers tolerance to salinity variances, the experiment was split into two parts: one is the pathway of the Artemia in varying salinities, and the second is the salinity tolerance in Artemia and dogwhelks ( Nucella lapillus ). Based upon the information known about osmoregulators and osmoconformers for the first part of the experiment of testing the Artemia pathway a null hypothesis was predicted as the Artemia will evenly disperse which means that they don’t have a preference for salinity. The first alternative hypothesis predicted was that the Artemia will go towards the higher salinity because that is the environment in which they are familiar with. The second alternative hypothesis predicted was that the Artemia will go towards the lower salinity because their bodies will not have to work as hard. For the second part of the experiment, a null hypothesis for the survival rate of the Artemia and dogwhelks to different salinities was predicted. The null hypothesis for Artemia was predicted as the Artemia will all die and show no preferences for salinity based on the fact that they are osmoregulators. The first alternative hypothesis was that the Artemia will

waters from mixing. The behavioral response was recorded indicating the direction the Artemia in that layer went. The experiment proceeded by testing the survival of Artemia and dogwhelks to differing salinities. 10 glass beakers were set up with seawater of different salinities: 0, 17, 35, and 50 ppt. Both species was then added to the varying treatments, 10 Artemia was added to each of the 5 beakers that were placed into a larger tub with the airstone placed in each to keep oxygen flowing into the beaker. The same was then repeated for 10 dogwhelks each being added to the 5 large glass beakers. A lid was placed on the beakers to prevent the dogwhelks from possibly moving out. At the end of day 1, 5 and 7 the number of alive organisms were counted. Data: Table 1: Artemia behavioral responses to 3 different salinity layers. Salinit y Level 0:30 1:00 1:30 2:00 2:30 3:00 3:30 4:00 4:30 5:00 17 ppt 0 -- -- -- 35 ppt -- -- -- -- 50 ppt 0 -- 0 -- -- -- -- 0 Table 2: Artemia : mean % of survival and standard deviation Salinity (ppt) Day 1 (Average % Survival) + SD Day 5 (Average % Survival) + SD Day 7 (Average % Survival) + SD 0 ppt 100 + 0 0 + 0 0 + 0 17 ppt 100 + 0 32.68 + 30.78 24.68 + 27.46 35 ppt 100 + 0 54.66 + 29.88 47.232 + 31.14 50 ppt 100 + 0 37.272 + 38.98 25.272 + 20.18

Table 3: Artemia : Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 5 0 0 0 Column 2 5 123.4 24.68 754.067 Column 3 5 236.16 47.232 969.39012 Column 4 5 126.36 25.272 407.16992 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 5587.27872 3 1862.42624 3.49648475 0.04017089 3.23887152 Within Groups 8522.50816 16 532.65676 Total 14109.7869 19 Graph 1: 0 ppt 17 ppt 35 ppt 50 ppt 0 20 40 60 80 100 120 % Survival of Artemia in different Salinity treatments Day 1 Day 5 Day 7 Salinity Treatment (ppt) % Survival Table 4: Nucella : dogwhelks: mean % of survival and standard deviation Salinity (ppt) Day 1 (Average % Survival) + SD Day 5 ( Average % Survival) + SD Day 7 (Average % Survival) + SD 0 ppt 100 + 0 0 + 0 0 + 0 17 ppt 100 + 0 98 + 4.47 76 + 21.91 35 ppt 100 + 0 49 + 0 38 + 21.91 50 ppt 100 + 0 78 + 23.88 52 + 28.64

observed that there was no movement over the next minute and thirty seconds. At the three- minute mark, the Artemia shifted to moving towards the lower salinity, while still remaining primarily in the intermediate and higher salinities. Thirty seconds later, there was no net movement observed in the layers until the fourth minute, when the Artemia moves in both lower and high salinity directions but mainly in the high salinity direction. By five minutes, there was no net change observed. Table 2 along with graph 1 displays the percent survival rate of the Artemia over the course of the 7 days within four different salinity variances. The lowest percent survival rate of Artemia was found in 0 ppt water. The Artemia began with an initial survival rate of 100 + 0% on day 1 and after 5 days, the Artemia percent survival was 0 + 0%, indicating no chance of survival for the Artemia in lowest salinity water. The second lowest percent survival rate is in 17 ppt water, beginning with an initial percent survival rate of 100 + 0%, and after 5 days the survival rate decreased to 32.68 + 30.78% and by day 7, the survival rate was 24.68 + 27.46%. The highest percent survival rate for the Artemia was found in 35 ppt water. The Artemia again began with an initial survival rate of 100 + 0% on day 1; however, after 5 days, unlike in 0 ppt and 17 ppt, the Artemia survival rate decreased to 54.66 + 29.88% percent survival rate, and on day 7 the survival rate was 47.20 + 31.14%. Tables 4 and graph 2, shows the percent survival rate for the dogwhelks ( Nucella lapillus ) over the course of the 7 days within the same four different salinity variances. Like the Artemia , the dogwhelks had zero percent rate of survival was in 0 ppt water. However, unlike the Artemia , the dogwhelks highest percent rate of survival was in 17 ppt, with an initial survival rate of 100 + 0%, and after 5 day, decreased to 98 + 4.47%. By day 7 the percent rate of survival had decreased to 76 + 21.91%.

Tables 3 and 5 show the results of an ANOVA test for both the Artemia and the dogwhelks ( Nucella lapillus ). An ANOVA: Single Factor test compares the survival rates within the different salinities for both species. Table 3 shows the ANOVA results for the Artemia , the F- value calculated was 3.49648475 and the F-critical was 3.23887152. Since the F-value is larger than the F-critical, the null hypothesis is rejected. This was confirmed by the ANOVA having a P-value of 0.04017089, which is less than the P-critical value of 0.05. This means that the results are significant, and the null hypothesis is rejected. Table 5 shows the ANOVA results for the dogwhelks, the F-value calculated was 16.5805243 and the F-critical was 3.23887152. The F- value for the dogwhelks was significantly higher than the F-critical, therefore, the null hypothesis is rejected. This was then confirmed by the ANOVA test showing a P-value of 3.6375*10 -05 , which is significantly less than the P-critical value of 0.05, meaning all the results are significant and the null hypothesis is rejected. Discussion: The purpose of this experiment was to get an understanding of the effects that salinity changes have on osmoregulators and osmoconformers. For the first portion of the experiment, the null hypothesis was that the Artemia would not have a preference for salinity. Upon examination, it was observed that the Artemia showed a preference for high and intermediate salinities because majority of the Artemia went towards the intermediate and high salinities as opposed to the few that moved towards the lower salinities. Artemia are raised in high salinity environments and their gills help them to deal with the high salt content by absorbing and excreting ions as necessary and producing a concentrated urine from the maxillary glands (ADW). Therefore, the experiment showed that they prefer to remain under the same high

than the Artemia . Unlike Artemia, the dogwhelks had the highest percentage of survival in the 17 ppt at 76 + 21.91% and the second highest at 50 ppt at 52 + 28.64%, showing that they can survive in wide ranges of salinities, however the intermediate salinities is preferred, proving the second hypothesis to be true. Compared to the Artemia, the dogwhelks had an overall higher percent survival rate because they are osmoconformers and are found on exposed shores and sheltered shores, and in sea lakes, estuaries, enclosed coasts, sounds and rias. They are found throughout the littoral zone and can survive under all types of tidal strength and wave exposure (Ocean Resources). As the experiment was conducted, time played a factor into the results of the experiment for both species. In both cases, the species survival rates declined over the course of the 7 days due to the time spent under stressful conditions. As for the rate of survival in Artemia in 35 ppt water and the dogwhelks ( Nucella lapillus ) in 17ppt water, conclusion can be drawn that the percent rate of survival decreased due to the quality of living conditions in the laboratory, such as proper filtering, limited feeding resources and limited spacing. From this information, it can be concluded that in the case of a large rainfall or flooding event in which the salinity of the water would be reduced, the dogwhelks are more likely to survive due to their ability to survive large salinity variations and high percent survivorship in 17 ppt water. Reference: “Dogwhelks, Nucella Lapillus.” Ocean Resources - MarineBio.org , 14 Jan. 2013, marinebio.org/species.asp?id=536. Emslie, Sara. “Artemia Salina.” Animal Diversity Web , 2003, animaldiversity.org/accounts/Artemia_salina/. Marine Biology 250, Summer 2010 Laboratory Assignment: Testing Taxis.

Nybakken, J.W., Bertness, M.D.2005. Marine Biology: An ecological approach. Pearson, California. Thurman, H.V., Trujillo, A.P. 2002. Essentials of Oceanography. 7th Ed. Prentice Hall, NJ. US Department of Commerce, and National Oceanic and Atmospheric Administration. “Estuaries, Adaptations to Life in the Estuary, NOS Education Offering.” NOAA's National Ocean Service , 19 Dec. 2005, oceanservice.noaa.gov/education/tutorial_estuaries/est07_adaptations.html.